CN1668744A - Method of transforming soybean - Google Patents
Method of transforming soybean Download PDFInfo
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- CN1668744A CN1668744A CN03816844.8A CN03816844A CN1668744A CN 1668744 A CN1668744 A CN 1668744A CN 03816844 A CN03816844 A CN 03816844A CN 1668744 A CN1668744 A CN 1668744A
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Abstract
The present disclosure provides methods for Agrobacterium-mediated transformation of soybean cells or tissue and regeneration of the transformed cells or tissue into transformed plants. The methods may be used for transforming many soybean cultivars.
Description
The cross reference of related application
The application requires the sequence number NO.60/390 of submission on June 22nd, 2002, the interests of 562 U.S. Provisional Application, and the full content of this application is incorporated this paper in the mode of reference.
Invention field
The present invention relates generally to the method for Plant Transformation, more specifically, relate to the method for soybean transformation cell and tissue.The invention still further relates to from the soya cells of conversion and the method for tissue regeneration genetically engineered soybean plant.The invention still further relates to the genetically engineered soybean Plants and Seeds that these methods obtain.
Background of invention
Soybean is a kind of main food and feed resource, and they are all more than any other dicotyledonous crops in soil of whole world plantation.Soybean planting surpasses 5,000 ten thousand hectares of areas according to reports.Unfortunately, the U.S. only the plant of minority introduce and to have produced the main cultivated variety of soybean, thereby the potential of soybean breeder has been limited on this narrow germplasm basis.This limited hereditary basis has limited with the traditional breeding way exploitation and has had the ability of the mutation of improvement or increase in value proterties in the soybean varieties of cultivation.
Therefore adopt gene engineering to modify the exploitation that soybean can promote new variety with traditional breeding way or the unapproachable mode of tissue culture induce variation, for example have the new variety of antiweed, disease-resistant (for example antiviral) and seed quality improved characteristics.
Developing effective conversion system is necessary for the analysis of genetic expression in the plant.The requirement of this system comprises suitable target plant tissue to allow effective plant regeneration, and the gene delivery means enters the target vegetable cell to transmit foreign DNA effectively, and effective means is to select cell transformed.In the genetic transformation of dicotyledonous kind, often use and utilize the conversion system of agrobacterium tumefaciens (Agrobacterium tumefaciens) as the gene delivery means.At present, the preferred target tissue that is used for agrobacterium mediation converted comprises cotyledon, leaf texture and hypocotyl.The high speed particle bombardment provides alternative method for gene is conveyed into dicotyledons.
To be far from be conventional to the transmission of agriculture bacillus mediated gene in the soybean.In the obtainable report of the public, often mention meristematic tissue and cotyledon tissue as the target that is used for agriculture bacillus mediated gene transmission.But, often can not reach reliable and effectively conversion and regeneration from these two explant resources.
The U.S. Patent number NO.5 of Chee etc., 169,770 and 5,376,543 have discussed the non-tissue culture method of soybean transformation generation transgenic plant, wherein cultivate seed germination, with bacterial cell particularly agrobacterium strains inoculation meristematic tissue or mesoblastic cell tissue, bacterial cell is transferred to DNA in the explant by infecting then.This method depends on the growth of preformed sprouting.
(1989) such as Parrot W.A., " recovery of the elementary transformant of soybean, " PlantCell Reports 7:615-617 has reported with Agrobacterium and has cultivated back recovery soybean transformant from immature cotyledon tissue altogether.But, these regenerated plants are chimeric, and metastatic gene can not entail filial generation.
U.S. Patent number NO.5,416,011 (Hinchee etc.) have discussed and have utilized the cotyledon explant that requires to remove hypocotyl, reservation and separate cotyledon, and inoculate this cotyledon explant to insert mosaic gene with the agrobacterium tumefaciens carrier that contains goal gene.
(2000) such as Yan B., " utilizing the Agrobacterium tumefaciens mediated soybean of prematurity zygote cotyledon explant to transform; " total transformation frequency was 0.03% during Plant Cell Reports 19:1090-1097, report transformed with the agriculture bacillus mediated soybean of unmature subleaf.
The U.S. Patent number NO.6 of Martinell etc., 384,301 have described agriculture bacillus mediated gene is conveyed in the isolating soybean plumular axis meristematic cell.These methods do not comprise callus phase tissue culture.
Work from the above description, apparently, when utilizing meristematic tissue and cotyledon tissue when being used for the resource explant of agriculture bacillus mediated gene transmission, relevant worker seldom realizes setting up the target of a reliable soybean conversion system.Therefore,, need to continue to explore new method, comprise new resource explant in order to develop more effective soybean conversion system.
Verified, in cultivating, soyabean tissue can obtain plant regeneration from epicotyl tissue and primary leaf tissue.But, up to now, the target that transmits as gene with these two kinds of explant sources in soybean does not also have the successfully report of conversion.
(1987) such as Wright M.S. " the initial sum breeding of Glycine max L.Merr.: from the epicotylar plant of tissue culture, " Plant Cell Reports 8:83-90, bud has been described from the successful initial sum propagation of soybean epicotyl tissue.In Schenk that contains 20 μ M phytokinin and Hildebrandt substratum, cultivate and induced the explant epicotyl to form bud in 5 weeks.Bud keeps propagation in the substratum that contains 2.1nM picloram and 0.1 μ M benzyladenine.
(1987) such as Wright M.S. " soybean (Glycine max L.Merr.) regenerates from the primary leaf tissue of cultivating, " Plant Cell Reports 6:83-89 has described the repeatably method of aftergrowth from the primary leaf tissue of 27 kinds of soybean.Although they find to have proved 2,4 that the 5-trichlorophenoxyacetic acid is necessary for regeneration, finds that benzyladenine (BA) can strengthen regeneration.
(1997) such as Rajasekaren K. " somatic embryo of the soybean of cultivation (Glycine max L.Merr) epicotyl and primary leaf takes place, " In Vitro Cellular ﹠amp; Developmental Biology 33 (2): 88-91, described several soybean and taken place from the cultivation epicotyl of the immature seed of greenhouse production plant and the regeneration of primary leaf tissue by somatic embryo.They find replenishing 46.2 μ M2 when the half son's leaf with complete zygote embryo axle, when cultivating on the Murashige of 4-D and Skoog (MS) substratum, can take place from epicotyl and primary leaf inductor cell stage.Do not having under the situation of cultivating, from the plumular axis, epicotyl or the primary leaf that extract, do not observing the embryo and take place with half son's leaf.On the ChengShi basic medium that contains 11.3 μ M benzyladenines, can obtain the fast breeding of bud point from the germination somatic embryo.
Summary of the invention
The invention provides the soybean transformation cell and transformant is regenerated as the method that transforms plant.Described method can be used for transforming multiple soybean culture kind.
The invention provides a kind of new soybean explant, its make agrobacterium tumefaciens efficiently mediated gene transfer in the soya cells.
Particularly, the invention provides the method for soybean transformation cell or tissue, described method comprises:
(a) prepare explant by following steps from soybean seeds:
(i) from said seed, remove all or part of hypocotyl;
(ii) from said seed, remove a cotyledon and contiguous axillalry bud (axillary bud) thereof, and keep one its go up the cotyledon of having epicotyl and primary leaf;
(iii) remove the part primary leaf that keeps cotyledon, produce the primary leaf base portion thus; And
(b) with this explant with contain the Agrobacterium that remains to be incorporated into the purpose nucleic acid in the soya cells genome and cultivate altogether.
In other embodiments, described method also comprises one or more following contents: form from primary leaf base portion and contiguous epicotyl induced bud thereof; In containing the substratum of selective agent, cultivate this bud; Select the bud of conversion; And shoot regeneration plant transformed from transforming.
In further embodiment, the invention provides the method for the soybean plants of production stable conversion, described method comprises:
(a) prepare explant by following steps from soybean seeds:
(i) from said seed, remove all or part of hypocotyl;
(ii) from said seed, remove a cotyledon and contiguous axillalry bud thereof, and keep one its go up the cotyledon of having epicotyl and primary leaf;
(iii) from epicotyl, remove the part primary leaf, produce at least one primary leaf base portion thus;
(b) cultivate this explant altogether and contain the Agrobacterium that remains to be incorporated into the genomic purpose nucleic acid of soya cells;
(c) form from primary leaf base area induced bud;
(d) in containing the substratum of selective agent, cultivate the bud that forms;
(e) bud of selection conversion; And
(f) the conversion bud selected of regeneration is a soybean plants.
In another embodiment, remove (a) (ii) part of middle each primary leaf of explant that produces, thereby produce a pair of primary leaf base portion.
Method of the present invention can be used for any purpose nucleic acid is imported in the soya cells.In one embodiment of the invention, described nucleic acid contains the gene of expressing required agronomy proterties in soybean.
In another embodiment of the present invention, described nucleic acid contains the Phophomannose isomerase gene as selectable marker gene.
In another embodiment of the present invention, explant and Agrobacterium cotransfection are to carry out under the situation that has seminose to exist.
Ripe and immature seed all can be used for producing the used explant of the present invention.
The accompanying drawing summary
Fig. 1 shows the collection of illustrative plates of plasmid pNOV2105.
Fig. 2 shows the collection of illustrative plates of plasmid pNOV2145.
Fig. 3 shows the collection of illustrative plates of plasmid pNOV2147.
Fig. 4 shows the demonstration methods of preparation soybean explant.Figure A has described the soybean seeds embryo, wherein removes a part of hypocotyl.Figure B has described the soybean explant from figure A, has wherein removed a cotyledon and contiguous axillalry bud thereof.Figure C has described the soybean explant of the figure B that removes two primary leafs, at the base portion generation breaking point of each primary leaf.
Fig. 5 shows the collection of illustrative plates of plasmid pBSC11234.
Fig. 6 shows the collection of illustrative plates of plasmid pBSC11369.
Detailed Description Of The Invention
Now, describe more all sidedly hereinafter the present invention with reference to accompanying drawing, wherein described multiple embodiments of the present invention. But, the present invention can embody in a different manner, and the embodiment shown in should not being interpreted as being limited to here. On the contrary, providing these embodiments is in order to make the disclosure more comprehensively with complete, to express all sidedly scope of the present invention to those skilled in the art. Here be used for term that the present invention describes and only be for specific embodiment is described without limits the present invention's meaning. In specification of the present invention and appended claim, unless outside clearly indicating in addition in the literary composition, singulative " " and " this " are intended to also comprise plural form.
What unless otherwise defined, technology used herein and scientific terminology and those skilled in the art understood usually is equivalent in meaning.
Unless otherwise indicated, but the Application standard method produce according to clone gene of the present invention, cell and the plant of expressing box, carrier (for example plasmid), albumen and protein fragments and transforming. Except as otherwise noted, but the Application standard method produce according to clone gene of the present invention, express box, carrier (for example plasmid), albumen and protein fragments. These technology are known to those skilled in the art, referring to for example, J.Sambrook etc., molecular cloning: laboratory manual second edition (cold spring harbor laboratory, Cold Spring Habor, New York, 1989), and F.M.Ausubel etc., current molecular biology experiment scheme (Green Publishing Associates, Inc. and Wiley-Interscienee, New York, 1991); The editors such as J.Draper, Genetic Transformation in Higher Plants and gene expression: laboratory manual, (Blackwell Scientific Publications, 1988); Edit the importing of gene outcome, expression and analysis in the plant with S.B.Gelvin ﹠ R.A. Schilperoort.
The invention describes the method and composition with purpose nucleotide sequence stable conversion soybean and regeneration of transgenic bean plant.
Method of the present invention is used in and expresses any purpose nucleic acid in the bean plant. Genes of interest can be, for example, herbicide gene, disease-resistant gene or anti-insect/insect gene, or selection or evaluation mark, and contain the exercisable promoter of plant, code area and 3 ' stops the subarea. The herbicide gene comprises to the AHAS gene of imidazolone or sulfonylurea herbicide resistance, to the pat of bialaphos or glufosinate resistance or bar gene, to epsp synthase gene of glyphosate resistance etc. Disease-resistant gene comprises the antibiotic synthase gene, pyrrolnitrin synthase gene for example, resistant gene of plant origin etc. The insect-resistant gene comprises Bactospeine bacillus insecticidal protein gene. The genes of interest enzyme relevant with bio-chemical pathway of also can encoding, the expression of this enzyme can change food, feed, nutritional drugs and/or medicine produce in important proterties. Genes of interest can be positioned on the plasmid. Being fit to plasmid that the present invention uses can contain an above genes of interest and/or Agrobacterium and can contain different plasmids with different genes of interest.
The invention provides the method for transformation of the various types of Glycine of the comprising max of Glycine (Soybean Species). Described method be take agriculture bacillus mediated genes of interest be conveyed into soya cells, the cytothesis that transforms afterwards is as the bean plant that transforms as the basis. Method of the present invention is independent of cultivar.
In one embodiment of the invention, explant is by sprouting a period of time the germination culture medium from soybean mature seed or immature seed that the greenhouse production plant is collected, remove kind of a skin from said mature seed or immature seed, remove afterwards cotyledon and prepare. In a preferred embodiment of the present invention, then remove the acrospire that a part exposes, thereby produce breakaway poing (Fig. 4) at acrospire base portion (primary leaf base). The cell of agriculture bacillus mediated gene delivery at acrospire base portion or acrospire breakpoint region carried out. Form from epicotylar acrospire base area evoking adventive bud (shoots). This is induced is the separate living tissue that pre-exists by removal (i.e. nascent, secondary and armpit give birth to separate living tissue), and explant is cultivated acquisition in containing the bud inducing culture of proper growth conditioning agent. Described bud abductive approach has promoted growth or the regeneration from the acrospire base portion transformation bud of target.
In the situation that selective agent exists, cultivate the soya cells that transforms. Preferably, phosphomannose isomerase (PMI) genetic transformation of these cells, and the gene that transforms is cultivated in the situation that mannose exists. In containing the culture medium that mannose is selective agent, the soya cells of PMI genetic transformation has growth vigor than the soya cells that does not have conversion like this.
Compare with other agriculture bacillus mediated method for transformation of reported in literature, the bean plant required time that transforms with the method for the invention regeneration has shortened significantly. The soybean transformation bud that begins to take root in 8 to 12 weeks from transformation experiment can generate. The exogenic heredity construct or the transgenosis that are inserted in the soybean gene group can be by conventional recombinant DNA operating technology in external structures. This construct can be comprised of any heterologous nucleic acids. This genetic constructs is transformed in the Agrobacterium strain in order to be transferred in the soya cells. Described Agrobacterium is non-carcinogenic, and several such bacterial strains can obtain at present widely. Described Agrobacterium is preferably selected from Agrobacterium tumefaciems (A.tumefaciens) and Agrobacterium rhizogenes (A.rhizogenes).
Preferably, the foreign gene construct contains selectable marker gene. Preferred selectable marker gene is Phophomannose isomerase gene. Other the suitable selectable marker gene gene of following substances: neomycin phosphotransferase II (Fraley etc., CRC Critical Reviews in Plant Science 4,1 (1986)) that includes, but not limited to encode; Cyanogen ammoniacal liquor synthase (Maier-Greiner etc., Proc.Natl.Acad.Sci.USA 88,4250 (1991)); Aspartokinase; Dihydrodipicolinate synthase (Perl etc., BioTechnology 11,715 (1993)); Bar gene (Toki etc., Plant Physiol.100,1503 (1992); Meagher etc., Crop Sci.36,1367 (1996)); Tryptophan decarboxylase (Goddijn etc., Plant Mol.Biol.22,907 (1993)); Neomycin phosphotransferase (NEO; Southern etc., J.Mol.Appl Gen.1,327 (1982)); Hygromix phosphotransferase (HPT or HYG; Shimizu etc., Mol.Cell.Biol.6,1074 (1986)); Dihyrofolate reductase (DHFR); Phosphinothricin acetyl transferase (DeBlock etc., EMBO J. 6,2513 (1987)); DPA dehalogenase (Buchanan-Wollatron etc., J.Cell.Biochem.13D, 330 (1989)); Acetohydroxy acid synthase (Anderson etc., U.S. Patent number No.4,761,373; Haughn etc., Mol.Gen.Genet.221,266 (1988)); 5-enol pyruvoyl-shikimic acid-phosphoric acid (5-enolpyruvyl-shikimate-phosphate) synthase (aroA; Comai etc., Nature 317,741 (1985)); Halogen aryl nitrile hydrolase (Stalker etc., WO 87/04181); Acetyl-CoA carboxylase (Parker etc., Plant Physiol.92,1220 (1990)); Dihydropteroate synthase (sul I; Guerineau etc., Plant Mol.Biol. 15,127 (1990)); And the Photosystem I I polypeptide (psbA of 32kDa; Hirschberg etc., Science 222,1346 (1983)).
The gene that also comprises has coding to the gene of following Drug-resistant: chloramphenicol (Herrera-Estrell etc., EMBO J.2,987 (1983)); Amethopterin (Herrera-Estrella etc., Nature 303,209 (1983); Meijer etc., Plant Mol.Biol.16,807 (1991)); Hygromycin (Waldron etc., Plant Mol.Biol. 5,103 (1985); Zhijian etc., Plant Science 108,219 (1995); Meijer etc., Plant Mol.Bio.16,807 (1991)); Streptomysin (Jones etc., Mol.Gen. Genet.210,86 (1987)); Spectinomycin (Bretagne-Sagnard etc., Transgenic Res.5,131 (1996)); Bleomycin (Hille etc., Plant Mol.Biol.7,171 (1986)); Sulfanilamide (SN) (Guerineau etc., Plant Mol.Bio.15,127 (1990)); Bromoxynil (Stalker etc., Science 242,419 (1988)); 2,4-D (Streber etc., Bio/Technology 7,811 (1989)); Phosphinothricin (DeBlock etc., EMBO J.6,2513 (1987)); Spectinomycin (Bretagne-Sagnard and Chupeau, Transgenic Research 5,131 (1996)).
In one embodiment, the parent material for method for transformation is the soybean mature seed. In another embodiment, parent material can be the soybean immature seed of the cultivated soybean plant. Described seed places the germination culture medium, allows its germination 6-24 hour, preferably germinates more preferably about 8-12 hour about 6-14 hour. If necessary, can also allow the germination longer time, for example 2 to 5 days.
Remove kind skin and the hypocotyl of chitting piece. Remove again a cotyledon and contiguous axillalry bud thereof. Then, acrospire is substantially removed, thereby produced an explant that contains the cotyledon that acrospire base portion, the basifixed epicotyl of this acrospire and this epicotyl adhere to. Substantially removing the meaning is the major part of having removed the acrospire tissue.
For agriculture bacillus mediated transgenosis, the wound of known plants tissue can promote transgenosis. Therefore, preferred but optional, can cause wound in the acrospire base regions.
Then, a few minutes in the explant immersion agrobatcerium cell suspension of method preparation as indicated above are arrived several hours, typically about 0.5-3 hour, preferably 1-2 hour. Remove too much agrobatcerium cell suspension, allow remaining Agrobacterium and explant in about 22 ℃ ± 2 ℃ temperature, on common culture medium, cultivated altogether several days under hour dark condition of illumination/8 in 16 hours, typically 2 to 5 days, preferably 3 to 4 days.
After being total to cultivation, explant is transferred to cultivation 8-12 week in the culture medium (or a series of culture medium), described culture medium is conducive to the selection of bud growth and transformant. Such culture medium (or these culture mediums) generally contains bud induces hormone and selective agent. The regeneration culture medium that uses among the following embodiment contains mannose as selective agent, and benayl aminopurine is induced hormone as bud. The term hormone also comprises the cell cycle regulation compound that induced bud forms, include but not limited to auximone (for example IAA, NAA and indolebutyric acid (IBA)), the basic element of cell division (cytokinins) (such as thidiazuron, kinetin (kinetin) and zeatin), and/or gibberellin (GA3)。
When bud is grown to about 2cm and had complete trilobal to become, bud is told from explant, and placed the formation of inducing root on the root media. Preferably, root media comprises that also selective agent is further to help to identify the bud that may transform. Root forms approximately needs 1-2 week, this plant can be transferred to afterwards and also cultivate full maturity in the soil.
Containing the genetically modified plants of heterologous nucleic acids (namely containing the cell or tissue that transforms according to this paper describing method) and seed and the offspring who produces by these genetically modified plants is another aspect of the present invention. The method of transformant being cultivated into useful cultivar is well known to a person skilled in the art. Whole plant regeneration technology is known in plant tissue Vitro Culture Techniques and the many situations. Another aspect of the present invention is genetically modified plants tissue, plant or the seed that contains above-mentioned nucleic acid. In preferred embodiments, the conversion of plant that uses methods described herein to produce is not chimeric, or the plant that only has sub-fraction to transform is chimeric. Preferably, this is by time of prolonging high concentration of cytokinin and processing or increase the stringency that mannose selects or both are all with realizing.
Therefore, by selection or Screening and Identification and after cultivating in the suitable culture medium of the support regeneration that provides such as this paper, can allow transformant of the present invention ripe is plant. Preferably, plant is ripe between cultivation or in the greenhouse. In about 2-6 week (depending on initial tissue) after transformant is identified, plant is regenerated. At regeneration period, cell can be cultivated by the solid medium in tissue culture vessel. The example embodiment of these culture vessels is culture dish and plant ConS. Plant in the regeneration is long to bud with after the root development stage, for further growth and test can be transferred to them in the greenhouse. Provide as mentioned, the seed of aftergrowth and progeny plants are one aspect of the present invention. Correspondingly, term " seed " means to comprise the seed of these conversion of plants and the seed that the conversion of plant offspring produces. Plant of the present invention not only comprises the plant that transforms and regenerate, and also comprises by the conversion of methods described herein generation and the offspring of aftergrowth.
Can from the plant that described method produces, screen successful conversion of plant by standard method as described above. For the Plants and Seeds strain (this also is another aspect of the present invention) of development and improvement, the seed of Selection and screening aftergrowth of the present invention and progeny plant are with the nucleotide sequence sustainable existence of render transgenic and integration constantly. Therefore, needed transgenosis nucleotide sequence can be moved in (namely gradually oozing or inbred) other genetic strain such as some original seed or commercial useful strain or kind. Gradually oozing the method that the purpose nucleotide sequence enters hereditary plant lines can realize by multiple technologies well known in the art, and these technology comprise by traditional breeding, protoplast fusion, nucleus and shifting and chromosome transfer. Breeding tactics is well known in the art, for example shown in the following document, and J.R.Welsh, plant genetic and breeding basis (John Wiley and Sons, New York, (1981)); Crop breeding (D.R.Wood, editor, American Society of Agronomy, Madison, Wisconsin, (1983)); O.Mayo, plant breeding is theoretical, second edition (Clarendon Press, Oxford, England (1987); And Wricke and Weber, Quantitative Genetics and selection plant breeding (Walter de Gruyter and Co., Berlin (1986)). Use other technology of these and this area, the genetically modified plants and the inbred strais that obtain according to the present invention can be used for producing commercial valuable hybrid plant and crops, and the kind of hybridization also is one aspect of the present invention.
Foregoing is illustrating of the various embodiments of the present invention, and should not regard as its restriction.
Further describe the present invention by the following examples, these embodiment also limit the scope of the invention never in any form.
Conversion carrier
Plasmid pNOV2105 (Fig. 1) is the modification to the pVictor of WO 97/04112 disclosure and description, wherein 35S promoter is replaced by the SMAS promotor, the 35S terminator is replaced by the Nos terminator, another SMAS promotor is inserted into the upstream of GUS intron GUS sequence, and GUS intron GUS sequence 3 ' terminal flank is the Nos terminator.The pNOV2105 that is used for methods described herein does not contain the multiple clone site of finding in pVictor.But, if necessary, increase such cloning site fully within the technology of this area.
PNOV2105 (Fig. 1) is the carrier that is used for agriculture bacillus mediated Plant Transformation, and contain and derive from nopaline type pTiT37 plasmid (Yadav etc., 1982 Proc Natl Acad Sci79:6322-6326) the right and left margin sequence of Ti, it is positioned at the flank of phosphomannose isomerase (PMI) and beta-Glucuronidase (GUS) gene.
For in intestinal bacteria (E.coli), duplicating and keeping, this plasmid contains replication origin (the pUC19ori) (Yanish-Perron etc. of escherichia coli plasmid pUC19,1985 Gene33:103-119), and in order to duplicate in agrobacterium tumefaciens and to keep, this plasmid also contains replication origin (pVS1ori) (the 1984 Plasmid 11:206-220 such as Itoh of pseudomonas (Pseudomonas) plasmid pVS1; Itoh and Hass 1985 Gene 36:27-36).For screening in intestinal bacteria and agrobacterium tumefaciens, this plasmid contains the coding 3 " spectinomycin/streptomycin resistance genes from transposon Tn7 of (9)-O-nucleotidyl transferase (spec/strep) (1985 Nucleic Acids Res 19:7095-7106 such as Fling).Be effectively expressing in bacterium, spec/strep resistant gene and tac promotor are (referring to as 1983 Gene 25 (203) such as Amann: 167-78) merge.
T-DNA fragment between the right side and left margin contains following gene, and these genes are to transfer to by Agrobacterium tumefaciens mediated conversion in the soybean plants gene only to be arranged.
GUS intron GUS
Beta-Glucuronidase (GUS): this fragment contains colibacillary beta-Glucuronidase gene by right margin, has an intron to translate to stop the Agrobacterium of merging SMAS promotor and Nos terminator in this coding region.GUS intron gus gene is isolating from plasmid pBISN1 (early transcription of agrobacterium dna 1986 tobaccos such as Narasimhulu and the corn, PlantCell 8:873-866).
Phosphomannose isomerase (PMI): this fragment is by left margin, be from colibacillary mannose-6-phosphate isomerase gene (Miles and Guest 1984, Gene 32:41-48), with SMAS promotor (NiM, Cui D, Einstein J, NarasimhuluS, Vergara CE, Gelvin SB (1995)) and the fusion of Nos terminator.Phophomannose isomerase gene as selective marker to contain on the substratum that the D-seminose is a carbon source screening transgenosis bud.
The composition of pNOV2145 (Fig. 2) and sequence are shown in SEQ ID NO:1.The composition of pNOV2147 (Fig. 3) and sequence are shown in SEQ ID NO:2.
Transform and regeneration
By release chlorine in moisture eliminator ripe exsiccant soybean seeds (Var.S42 H1) is carried out surface sterilization.Seed is kept in the petri culture dish, and chlorine is by pouring 100ml Clorox in beaker
And slowly add the dense HCL of 8ml and produce.Seed discharges to handle with twice chlorine at least sterilizes, and continues 8-18 hour at every turn.
Then, disinfectant seed (the about 15-20 of an every ware seed) is placed MS basic medium (the modification substratum of quick growth of Murashige and Skoog (1962) tobacco healing tissue culture and biochemical identification that contains 0.6% agar and solidify.Physiol Plant 15:473-479) and on the germination substratum of 2% sucrose.The pH value keeps 5.8.The petri culture dish places 37 ℃ of rooms to make seed overnight growth or imbibition.Remove kind of a skin, remove the part hypocotyl then, keep the hypocotyl of about 0.5cm.Remove a cotyledon together with its contiguous axillalry bud and abandon.On remaining cotyledon, disconnect primary leaf with scalpel, keep the primary leaf base portion (Fig. 4) on the epicotyl.
Contain plasmid pNOV2145 (ZsGreen1 and PMI with what preserve in the frozen glycerine storing solution, as described in embodiment 1) agrobacterium strains (LBA 4404) line at YEP (the yeast extract 10g/L that contains suitable microbiotic (100mg/L spectinomycin), peptone 5g/L, sodium-chlor 5g/L, Bacto-agar 15g/L) on the culture dish.Then, with 27 ℃ of incubation 1-2 of Agrobacterium days.Get one spoonful of Agrobacterium from culture dish and put into YEP liquid nutrient medium 27 ℃ of overnight incubation on shaking table that 100ml contains microbiotic (100mg/L spectinomycin).Bacterial suspension is centrifugal 15 minutes of about 1500g, is resuspended in then in the common cultivation liquid nutrient medium to reach density OD
660=0.2 or 0.65, the consisting of of this substratum: B
5Salt 0.05 * (Sigma), B
5VITAMIN (0.05 *) (B
5Vitamin composition (1 *): inositol 100mg/L, nicotinic acid 1mg/L, pyridoxine hydrochloride 1mg/L, vitamin 10mg/L), Syringylethanone 40mg/L, sucrose 20g/L, BAP 2mg/L, GA
30.25mg/L, MES (morpholino ethane sulfonic acid) 3.9g/L and pH5.4.
The explant that will contain target tissue immerses in the agrobacterium suspension, and hatches 1-2 hour.Outwell the Agrobacterium suspension, then the explant of handling is placed on the filter paper of common cultivation culture dish.Keep the paraxial side of this explant to contact with filter paper.Cultivate solid medium altogether by B
5Salt (Sigma, 0.05 *), B
5VITAMIN (0.05 *), 40mg/L Syringylethanone, sucrose 20g/L, BAP 2mg/L, GA
30.25mg/L, MES 3.9g/L and pH5.4 form.This substratum solidifies with 0.5% refining agar (Sigma).
Explant and Agrobacterium were cultivated 2-5 days at 20-30 ℃ under hour dark condition of illumination/8 altogether at 16 hours.After cultivating altogether, rinsing explant in the sterilized water that contains 250mg/L cefotaxime cephamycin is removed nascent and secondary meristem, then explant is transferred in the regeneration culture medium (being the REG-1 substratum).In regenerative process, the armpit of also removing contiguous cotyledon is sprouted to promote the growth of primary leaf base area.
The REG-1 substratum contains MS salt (1 *), B
5VITAMIN (1 *), KNO
31g/L, BAP 1mg/L, ticarcillin (ticarcillin) 300mg/L, cefotaxime cephamycin 100mg/L, glutamine 250mg/L, l-asparagine 50mg/L, seminose 15-30g/L, sucrose 0,0.25 and 1g/L, pH5.6 and refining agar 10g/L.In each petri culture dish, place five explants, insert in the substratum so that the epicotyl of explant is terminal with orthostatism.Culture dish is placed in the plastic containers, and in being placed between 22-25 ℃ cultivation, at every 18-20 hour illumination/4-6 hour dark cycle, light intensity 60-100 μ Em
-2S
-1Condition under cultivate.After 2 weeks of growth on the REG-1 substratum, explant to be transferred on the REG-2 substratum, the REG-2 substratum contains MS salt (1 *) and B
5VITAMIN (1 *), KNO
31g/L, BAP 0.5mg/L, ticarcillin 300mg/L, cefotaxime cephamycin 100mg/L, glutamine 250mg/L, l-asparagine 50mg/L, seminose 15g/L and sucrose 1g/L.The pH value of substratum remains on 5.6, and substratum solidifies with refining agar 10g/L.
In 4-6 week, the soybean culture is transferred to continuation selection and bud formation on the REG-3 substratum.The REG-3 substratum contains MS salt (1 *), B
5VITAMIN (1 *), KNO
31g/L, BAP 0.2mg/L, GA
30.5mg/L, IBA 0.1mg/L, ticarcillin 300mg/L, cefotaxime cephamycin 100mg/L, glutamine 250mg/L, l-asparagine 50mg/L, seminose 15g/L, sucrose 1g/L, pH5.6, substratum solidifies with refining agar 10g/L.Remove dead tissue, have per two weeks of the explant of regeneration bud with the cultivation of going down to posterity of fresh REG-3 substratum.When bud length is long to about 2-4cm, gather in the crops the bud that extends from culture continuously.Simultaneously, bud is transferred in the root media, root media contains MS salt (0.5 *), B
5VITAMIN (0.5 *), glutamine 250mg/L, l-asparagine 50mg/L, KNO
31g/L, cefotaxime cephamycin 100mg/L, ticarcillin 300mg/L, sucrose 15g/L, IBA 0.5mg/L, pH5.6 and refining agar 10g/L.
The transgenosis bud of taking root of express fluorescent protein gene (ZsGreen1) transferred to contain moistening Fafard germination mixture (Conrad Fadard Inc., MA in 211 culture tank USA), and cover to preserve moisture with plastic cup and continue to cultivate about 2 weeks.Temperature 27-29 ℃ by day of plant, 21 ℃, 16 hours periodicity of illumination (light intensity 20-40 μ Em of nocturnal temperature
-2S
-1) condition under tame.When young leaves begins to grow, plant is changed in the one gallon of culture tank that contains soil mixture, (Sungrow Horticultural Supply, Pine Bluff Arkansas) form this soil mixture by 50-55% pine tree bark compost, 40-45% peat, 5-10% perlite.The soybean plants of domestication day temperature 27-29 ℃, 21 ℃ of nocturnal temperatures, light intensity 400-600 μ Em in the greenhouse
-2S
-1, relative humidity 70-95% and 16 hours periodicity of illuminations condition under grow.These plants are at growing period osmocote (Scotts-SierraHorticultural Products Company, Ohio; 17-6-12) twice (5-8g/ gallon soil) of fertilising.Analyze the existence that fluorescence protein gene and PMI gene are arranged by Taqman in the greenhouse production plant leaf and confirm that the generation of conversion is arranged.By using fluorescent microscope to manifest the expression that expression has also confirmed fluorescence protein gene in the soyabean tissue that transforms.
Confirmed to have obtained six transgenic plant by the Southern engram analysis with gene construct pNOV2145.In the genome that the offspring analysis PMI gene or the ZsGreen1 gene of an incident is disclosed in soybean transformation a T-DNA integration site is arranged.This filial generation separates with the 3:1 ratio in generation at T1.
The bud of the express fluorescent protein gene (ZsGreen1) that table 1 transforms
| Experiment numbers | Gene construct | Bud/the explant that transforms | Transform bud per-cent |
| ????75 | ??pNOV2145 | ??????5/45 | ??????11 |
| ????89 | ??pNOV2145 | ??????5/75 | ??????7 |
Embodiment 3
With soybean seeds (Var.S42 H1) surface sterilization, and as preparation explant as described in the embodiment 2.
The agrobacterium strains (LBA4404) that contains plasmid pNOV2147 as preparation as described in the embodiment 1.With cultivating liquid nutrient medium altogether the bacterium final concentration is adjusted into OD
660=0.60.Explant preparation, Agrobacterium inoculate and culture condition is described identical with embodiment 2 altogether.
After in the common culture medium of solid, cultivating three days altogether, too much Agrobacterium is rinsed, remove nascent and secondary meristem, and explant is transferred in the REG-1 substratum.They are cultivated under 28-30 ℃ of 16 little time/8 hour dark conditions.After cultivating for 2 weeks on the REG-1 substratum, culture is transferred in the REG-2 substratum.In this regenerative process, only keep the bud that bears from the primary leaf base portion.Around about, the bud culture is transferred in the REG-3 substratum.Then, transferred in the fresh REG-3 substratum in every 10-14 days.The same at REG-1 and REG-2 substratum, only keep the bud that produces from the primary leaf base portion and remove all the other buds.When the bud length of extending reaches about 2-4cm, they and the other parts of bud culture are separated, and transfer in the root media.
From 35 explants, identify five transgenosis buds (table 2) of expressing blue fluorescence protein gene.
The bud of blue fluorescin (the cyano fluorescent protein) gene of the expression that table 2 transforms.
| Experiment numbers | Gene construct | Bud/the explant that transforms | The bud that % transforms |
| ????92 | ???pNOV2147 | ??????5/35 | ?????14 |
Embodiment 4
Seminose between incubation period is handled altogether
Used in the present embodiment gene construct is that (it contains ZsGreen1 and PMI gene to pNOV2145, as described in example 1 above).Method the carrying out as described in example 2 above for preparing explant, Agrobacterium suspension and bacterial suspension inoculation explant.The bacterium final concentration is adjusted into OD
660=0.55 or 0.85.
After the inoculation step, explant is transferred in the common culture medium that contains 20g/L sucrose or 15g/L seminose, and remained on cultivation under the 20-23 ℃ of 16 little time/8 hour dark conditions.
After cultivating 3-5 days altogether, use the expression of fluorescence microscope fluorescence protein gene.The explant of inoculation Agrobacterium in containing the common culture medium of seminose is compared with the explant of the Agrobacterium inoculation of cultivating altogether in the common culture medium that contains sucrose, shows that the fluorescent spot of twice is counted at least.Shoot regeneration afterwards and selection step method are as described in example 2 above carried out.
Contain in culture medium altogether and to find in the experiment of seminose that the output that transforms bud has increased (table 3) significantly.The blastogenesis root of five conversions is arranged from the common culture medium that contains seminose, and transferred in the soil.Taqman and Southern engram analysis confirm to have genetically modified integration afterwards.The kind system that has confirmed these metastatic genes in T1 filial generation transfer expression of gene transmits.
Table 3 is expressed the conversion bud of ZsGreen1 fluorescence protein gene, and wherein explant and Agrobacterium are cultivated in seminose or sucrose altogether
| Experiment numbers | Gene construct | In seminose or sucrose, cultivate altogether | Bud/the explant that transforms | % transforms |
| ????87 | ??pNOV2145 | The sucrose seminose | ??????0/60 ??????6/80 | ????0 ????7.5 |
| ????102 | ??pNOV2145 | The sucrose seminose | ??????1/20 ??????8/40 | ????5 ????20 |
Embodiment 5
In the present embodiment, use and to contain plasmid pNOV2105 (SMAS-PMI SMAS-GUS, Agrobacterium EHA101 as described in example 1 above) are used for soybean and transform.Identical with described in the embodiment 2 of the preparation of explant, Agrobacterium suspension and Agrobacterium inoculation explant.The bacterium final concentration is adjusted into OD
660=0.45 or 0.6.
After the Agrobacterium inoculation, explant is transferred in the common culture medium that contains 20g/L sucrose or 15g/L seminose.Under 20-23 ℃ of 16 little time/8 hour dark conditions, carry out common cultivation.After cultivating 3-5 days altogether, observe the expression of gus gene with the gus of histological chemistry measuring method.The explant of cultivating altogether in containing the common culture medium of seminose is compared with the explant of cultivating altogether in containing the common culture medium of sucrose, shows the GUS spot number of twice at least.Shoot regeneration and select as described in example 2 above method to carry out.In being total to culture medium, observe the output that transforms bud in the experiment of adding seminose and significantly increase (table 4).
Table 4 is expressed the conversion bud of gus gene
| Experiment numbers | Gene construct | In seminose or sucrose, cultivate altogether | Bud/the explant that transforms | % transforms |
| ????63 | ??pNOV2105 | Sucrose | ?????5/60 | ????8 |
| ????81 | ??pNOV2105 | The sucrose seminose | ?????2/30 ?????5/30 | ????7 ????17 |
Embodiment 6
In the present embodiment, using the Agrobacterium EHA101 that contains plasmid pBSC11234 (Fig. 5) to be used for soybean transforms.The composition of pBSC11234 and sequence are shown in SEQ ID NO:3.PBSC11234 contains CMP-PMI: beta-conglycinin (conglycinin)-galactosidase gene construct.Identical with described in the embodiment 2 of the preparation of explant, Agrobacterium suspension and Agrobacterium inoculation explant.The bacterium final concentration is adjusted into OD
660=0.6.This is cultivated liquid nutrient medium altogether and contains B
5Salt (0.1 *), B
5VITAMIN (1 *), Syringylethanone 80mg/L, sucrose 20g/L, BAP 2mg/L, GA
30.25mg/L, MES 3.9g/L and pH5.4.Prepare solid culture medium altogether by in the common culture medium of liquid, mixing the refining agar of 5g/L.
After the Agrobacterium inoculation, explant is transferred to solid be total on the culture medium, under 20-24 ℃ of 16 little time/8 hour dark conditions, cultivate.After cultivating 3-5 days altogether, remove nascent and the meristematic tissue and abandoning of time sprouting, resulting explant is transferred in the REG-4 substratum.The REG-4 substratum contains B
5Salt (1 *), B
5VITAMIN (1 *), BAP 1mg/L, glutamine 50mg/L, l-asparagine 50mg/L, cefotaxime cephamycin 100mg/L, ticarcillin 300mg/L, seminose 15-20g/L, sucrose 0,0.25 or 1g/L, refining agar 10g/L and pH5.6.After during 5-7 days, remove any bud that grows from the axillary meristem near cotyledon, explant is transferred in the REG-5 substratum then, the REG-5 substratum contains B
5Salt (1 *), B
5VITAMIN (1 *), BAP 0.5mg/L, glutamine 50mg/L, l-asparagine 50mg/L, cefotaxime cephamycin 100mg/L, ticarcillin 300mg/L, seminose 15-20g/L, sucrose 1g/L, refining agar 10g/L and pH5.6.All around the time, explant is transferred in the REG-6 substratum so that the bud elongation.The REG-6 substratum contains MS salt (1 *), MS VITAMIN (1 *) (MS vitamin composition: inositol 100mg/L, nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, vitamin 0.1mg/L, glycine 2mg/L), inositol 200mg/L, BAP 0.2mg/L, zeatin ribonucleoside 0.5mg/L, IBA 0.1mg/L, GA
31mg/L, glutamine 50mg/L, l-asparagine 50mg/L, ticarcillin 300mg/L, seminose 15g/L, sucrose 5g/L, Silver Nitrate 0.8mg/L, refining agar 10g/L and pH5.6.Per two weeks are transferred to explant in the fresh REG-6 substratum.The bud (2-4cm is long) of elongation is shifted out, and in root media, take root, transfer in the soil then.Root media contains MS salt (1 *), B
5VITAMIN (1 *), glutamine 100mg/L, l-asparagine 100mg/L, IBA 0.7mg/L, timentin 100mg/L and sucrose 15g/L.Taqman analyzes the existence that confirms to have transgenosis (α tilactase and phosphomannose isomerase) in the leaf sample of two incidents.
Embodiment 7
In the present embodiment, using the Agrobacterium EHA101 that contains plasmid pBSC11369 (Fig. 6) to be used for soybean transforms.The composition of pBSC11369 and sequence are shown in SEQ ID NO:4.PBSC11369 contains the CMP-HPT:CMP-ZsGreen1 gene construct.Identical with described in the embodiment 2 of the preparation of explant, Agrobacterium suspension and Agrobacterium inoculation explant.The bacterium final concentration is adjusted into OD
660=0.6.The described liquid nutrient medium of cultivating altogether contains B
5Salt (0.1 *), B
5VITAMIN (1 *), Syringylethanone 80mg/L, sucrose 20g/L, BAP 2mg/L, GA
30.25mg/L, MES 3.9g/L and pH5.4.Prepare solid culture medium altogether by in the common culture medium of liquid, mixing the refining agar of 5g/L.
After the Agrobacterium inoculation, explant is transferred to solid be total on the culture medium, under 20-24 ℃ of 16 little time/8 hour dark conditions, cultivate.After cultivating 3-5 days altogether,, remove nascent and secondary meristem, afterwards explant is transferred in the REG-7 substratum from explant for promoting bud from the growth of primary leaf base area.The REG-7 substratum contains B
5Salt (1 *), B
5VITAMIN (1 *), BAP 1mg/L, glutamine 50mg/L, l-asparagine 50mg/L, cefotaxime cephamycin 100mg/L, ticarcillin 300mg/L, sucrose 30g/L, Totomycin 2-5mg/L, refining agar 10g/L and pH5.6.Explant is uprightly placed so that in the epicotyl end insertion substratum with explant.After during 7-10 days, remove all buds that grow from axillary meristem near cotyledon.Explant is transferred in the fresh REG-8 substratum, and the REG-8 substratum contains B
5Salt (1 *), B
5VITAMIN (1 *), BAP 0.5mg/L, glutamine 50mg/L, l-asparagine 50mg/L, cefotaxime cephamycin 100mg/L, ticarcillin 300mg/L, sucrose 30g/L, refining agar 10g/L and pH are 5.6.After two weeks, explant is transferred in the REG-9 substratum cultivation of going down to posterity in per then two weeks again.The REG-9 substratum contains MS salt (1 *), MS VITAMIN (1 *), inositol 200mg/L, BAP 0.2mg/L, zeatin ribonucleoside 0.5mg/L, IBA 0.1mg/L, GA
31mg/L, glutamine 50mg/L, l-asparagine 50mg/L, Silver Nitrate 0.8mg/L, ticarcillin 300mg/L, sucrose 30g/L, Totomycin 0.1-0.2mg/L, refining agar 10g/L and pH5.6.The bud (2-4cm is long) of elongation is shifted out, in root media, take root, transfer in the soil then.Root media contains MS salt (1 *), B
5VITAMIN (1 *), glutamine 100mg/L, l-asparagine 100mg/L, IBA 0.7mg/L, timentin 100mg/L and sucrose 15g/L.Taqman analyzes the existence that confirms to have metastatic gene (HPT and ZsGreen1) from the leaf sample that five incidents obtain.Confirmed that by under fluorescent microscope, observing the ZsGreen1 expression of gene is arranged in plant part.
This paper is incorporated in all publications, patent and patent application that this paper quotes as a reference into.Although in the specification sheets in front, described some relevant preferred embodiment of the present invention and for example explanation stated many details, but what it will be apparent to those skilled in the art that is the present invention can have other embodiments and some details described herein sizable variation can be arranged and do not depart from fundamental principle of the present invention.
Sequence table
<110>Syngenta?Participations?AG
＜120〉method of soybean transformation
<130>70094?USPS
<160>4
<170>PatentIn?version?3.2
<210>1
<211>9555
<212>DNA
＜213〉artificial
<220>
<223>pNOV2145
<400>1
gatccaccgg?tcgccaccat?ggcccagtcc?aagcacggcc?tgaccaagga?gatgaccatg?????60
aagtaccgca?tggagggctg?cgtggacggc?cacaagttcg?tgatcaccgg?cgagggcatc????120
ggctacccct?tcaagggcaa?gcaggccatc?aacctgtgcg?tggtggaggg?cggccccttg????180
cccttcgccg?aggacatctt?gtccgccgcc?ttcatgtacg?gcaaccgcgt?gttcaccgag????240
tacccccagg?acatcgtcga?ctacttcaag?aactcctgcc?ccgccggcta?cacctgggac????300
cgctccttcc?tgttcgagga?cggcgccgtg?tgcatctgca?acgccgacat?caccgtgagc????360
gtggaggaga?actgcatgta?ccacgagtcc?aagttctacg?gcgtgaactt?ccccgccgac????420
ggccccgtga?tgaagaagat?gaccgacaac?tgggagccct?cctgcgagaa?gatcatcccc????480
gtgcccaagc?agggcatctt?gaagggcgac?gtgagcatgt?acctgctgct?gaaggacggt????540
ggccgcttgc?gctgccagtt?cgacaccgtg?tacaaggcca?agtccgtgcc?ccgcaagatg????600
cccgactggc?acttcatcca?gcacaagctg?acccgcgagg?accgcagcga?cgccaagaac????660
cagaagtggc?acctgaccga?gcacgccatc?gcctccggct?ccgccttgcc?ctgagcggcc????720
ctctagatcc?ccgaatttcc?ccgatcgttc?aaacatttgg?caataaagtt?tcttaagatt????780
gaatcctgtt?gccggtcttg?cgatgattat?catataattt?ctgttgaatt?acgttaagca????840
tgtaataatt?aacatgtaat?gcatgacgtt?atttatgaga?tgggttttta?tgattagagt????900
cccgcaatta?tacatttaat?acgcgataga?aaacaaaata?tagcgcgcaa?actaggataa????960
attatcgcgc?gcggtgtcat?ctatgttact?agatcgggaa?ttgggtaccg?aattcactgg???1020
ccgtcgtttt?acaacgtcgt?gactgggaaa?accctggcgt?tacccaactt?aatcgccttg???1080
cagcacatcc?ccctttcgcc?agctggcgta?atagcgaaga?ggcccgcacc?gatcgccctt???1140
cccaacagtt?gcgcagcctg?aatggcgaat?ggcgcctgat?gcggtatttt?ctccttacgc???1200
atctgtgcgg?tatttcacac?cgcatatggt?gcactctcag?tacaatctgc?tctgatgccg???1260
catagttaag?ccagccccga?cacccgccaa?cacccgctga?cgcgccctga?cgggcttgtc????1320
tgctcccggc?atccgcttac?agacaagctg?tgaccgtctc?cgggagctgc?atgtgtcaga????1380
ggttttcacc?gtcatcaccg?aaacgcgcga?gacgaaaggg?cctcgtgata?cgcctatttt????1440
tataggttaa?tgtcatgata?ataatggttt?cttagacgtc?aggtggcact?tttcggggaa????1500
atgtgcgcgg?aacccctatt?tgtttatttt?tctaaataca?ttcaaatatg?tatccgctca????1560
tgagacaata?accctgataa?atgcttcaat?ggcgcgccgg?taccagcttg?catgcctgca????1620
ggtcgactct?agaggatcct?ggcagacaaa?gtggcagaca?tactgtccca?caaatgaaga????1680
tggaatctgt?aaaagaaaac?gcgtgaaata?atgcgtctga?caaaggttag?gtcggctgcc????1740
tttaatcaat?accaaagtgg?tccctaccac?gatggaaaaa?ctgtgcagtc?ggtttggctt????1800
tttctgacga?acaaataaga?ttcgtggccg?acaggtgggg?gtccaccatg?tgaaggcatc????1860
ttcagactcc?aataatggag?caatgacgta?agggcttacg?aaataagtaa?gggtagtttg????1920
ggaaatgtcc?actcacccgt?cagtctataa?atacttagcc?cctccctcat?tgttaaggga????1980
gcaaaatctc?agagagatag?tcctagagag?agaaagagag?caagtagcct?agaagtagga????2040
tccccgatca?tgcaaaaact?cattaactca?gtgcaaaact?atgcctgggg?cagcaaaacg????2100
gcgttgactg?aactttatgg?tatggaaaat?ccgtccagcc?agccgatggc?cgagctgtgg????2160
atgggcgcac?atccgaaaag?cagttcacga?gtgcagaatg?ccgccggaga?tatcgtttca????2220
ctgcgtgatg?tgattgagag?tgataaatcg?actctgctcg?gagaggccgt?tgccaaacgc????2280
tttggcgaac?tgcctttcct?gttcaaagta?ttatgcgcag?cacagccact?ctccattcag????2340
gttcatccaa?acaaacacaa?ttctgaaatc?ggttttgcca?aagaaaatgc?cgcaggtatc????2400
ccgatggatg?ccgccgagcg?taactataaa?gatcctaacc?acaagccgga?gctggttttt????2460
gcgctgacgc?ctttccttgc?gatgaacgcg?tttcgtgaat?tttccgagat?tgtctcccta????2520
ctccagccgg?tcgcaggtgc?acatccggcg?attgctcact?ttttacaaca?gcctgatgcc????2580
gaacgtttaa?gcgaactgtt?cgccagcctg?ttgaatatgc?agggtgaaga?aaaatcccgc????2640
gcgctggcga?ttttaaaatc?ggccctcgat?agccagcagg?gtgaaccgtg?gcaaacgatt????2700
cgtttaattt?ctgaatttta?cccggaagac?agcggtctgt?tctccccgct?attgctgaat????2760
gtggtgaaat?tgaaccctgg?cgaagcgatg?ttcctgttcg?ctgaaacacc?gcacgcttac????2820
ctgcaaggcg?tggcgctgga?agtgatggca?aactccgata?acgtgctgcg?tgcgggtctg????2880
acgcctaaat?acattgatat?tccggaactg?gttgccaatg?tgaaattcga?agccaaaccg????2940
gctaaccagt?tgttgaccca?gccggtgaaa?caaggtgcag?aactggactt?cccgattcca????3000
gtggatgatt?ttgccttctc?gctgcatgac?cttagtgata?aagaaaccac?cattagccag????3060
cagagtgccg?ccattttgtt?ctgcgtcgaa?ggcgatgcaa?cgttgtggaa?aggttctcag????3120
cagttacagc?ttaaaccggg?tgaatcagcg?tttattgccg?ccaacgaatc?accggtgact????3180
gtcaaaggcc?acggccgttt?agcgcgtgtt?tacaacaagc?tgtaagagct?tactgaaaaa????3240
attaacatct?cttgctaagc?tgggagctct?agatccccga?atttccccga?tcgttcaaac????3300
atttggcaat?aaagtttctt?aagattgaat?cctgttgccg?gtcttgcgat?gattatcata????3360
taatttctgt?tgaattacgt?taagcatgta?ataattaaca?tgtaatgcat?gacgttattt????3420
atgagatggg?tttttatgat?tagagtcccg?caattataca?tttaatacgc?gatagaaaac????3480
aaaatatagc?gcgcaaacta?ggataaatta?tcgcgcgcgg?tgtcatctat?gttactagat????3540
cgggaattgg?gtaccatgcc?cgggcggcca?gcatggccgt?atccgcaatg?tgttattaag????3600
ttgtctaagc?gtcaatttgt?ttacaccaca?atatatcctg?ccaccagcca?gccaacagct????3660
ccccgaccgg?cagctcggca?caaaatcacc?actcgataca?ggcagcccat?cagaattaat????3720
tctcatgttt?gacagcttat?catcgactgc?acggtgcacc?aatgcttctg?gcgtcaggca????3780
gccatcggaa?gctgtggtat?ggctgtgcag?gtcgtaaatc?actgcataat?tcgtgtcgct????3840
caaggcgcac?tcccgttctg?gataatgttt?tttgcgccga?catcataacg?gttctggcaa????3900
atattctgaa?atgagctgtt?gacaattaat?catccggctc?gtataatgtg?tggaattgtg????3960
agcggataac?aatttcacac?aggaaacaga?ccatgaggga?agcgttgatc?gccgaagtat????4020
cgactcaact?atcagaggta?gttggcgtca?tcgagcgcca?tctcgaaccg?acgttgctgg????4080
ccgtacattt?gtacggctcc?gcagtggatg?gcggcctgaa?gccacacagt?gatattgatt????4140
tgctggttac?ggtgaccgta?aggcttgatg?aaacaacgcg?gcgagctttg?atcaacgacc????4200
ttttggaaac?ttcggcttcc?cctggagaga?gcgagattct?ccgcgctgta?gaagtcacca????4260
ttgttgtgca?cgacgacatc?attccgtggc?gttatccagc?taagcgcgaa?ctgcaatttg????4320
gagaatggca?gcgcaatgac?attcttgcag?gtatcttcga?gccagccacg?atcgacattg????4380
atctggctat?cttgctgaca?aaagcaagag?aacatagcgt?tgccttggta?ggtccagcgg????4440
cggaggaact?ctttgatccg?gttcctgaac?aggatctatt?tgaggcgcta?aatgaaacct????4500
taacgctatg?gaactcgccg?cccgactggg?ctggcgatga?gcgaaatgta?gtgcttacgt????4560
tgtcccgcat?ttggtacagc?gcagtaaccg?gcaaaatcgc?gccgaaggat?gtcgctgccg????4620
actgggcaat?ggagcgcctg?ccggcccagt?atcagcccgt?catacttgaa?gctaggcagg????4680
cttatcttgg?acaagaagat?cgcttggcct?cgcgcgcaga?tcagttggaa?gaatttgttc????4740
actacgtgaa?aggcgagatc?accaaagtag?tcggcaaata?aagctctagt?ggatctccgt????4800
acccccgggg?gatctggctc?gcggcggacg?cacgacgccg?gggcgagacc?ataggcgatc????4860
tcctaaatca?atagtagctg?taacctcgaa?gcgtttcact?tgtaacaacg?attgagaatt????4920
tttgtcataa?aattgaaata?cttggttcgc?atttttgtca?tccgcggtca?gccgcaattc????4980
tgacgaactg?cccatttagc?tggagatgat?tgtacatcct?tcacgtgaaa?atttctcaag????5040
cgctgtgaac?aagggttcag?attttagatt?gaaaggtgag?ccgttgaaac?acgttcttct????5100
tgtcgatgac?gacgtcgcta?tgcggcatct?tattattgaa?taccttacga?tccacgcctt????5160
caaagtgacc?gcggtagccg?acagcaccca?gttcacaaga?gtactctctt?ccgcgacggt????5220
cgatgtcgtg?gttgttgatc?taaatttagg?tcgtgaagat?gggctcgaga?tcgttcgtaa????5280
tctggcggca?aagtctgata?ttccaatcat?aattatcagt?ggcgaccgcc?ttgaggagac????5340
ggataaagtt?gttgcactcg?agctaggagc?aagtgatttt?atcgctaagc?cgttcagtat????5400
cagagagttt?ctagcacgca?ttcgggttgc?cttgcgcgtg?cgccccaacg?ttgtccgctc????5460
caaagaccga?cggtcttttt?gttttactga?ctggacactt?aatctcaggc?aacgtcgctt????5520
gatgtccgaa?gctggcggtg?aggtgaaact?tacggcaggt?gagttcaatc?ttctcctcgc????5580
gtttttagag?aaaccccgcg?acgttctatc?gcgcgagcaa?cttctcattg?ccagtcgagt????5640
acgcgacgag?gaggtttatg?acaggagtat?agatgttctc?attttgaggc?tgcgccgcaa????5700
acttgaggca?gatccgtcaa?gccctcaact?gataaaaaca?gcaagaggtg?ccggttattt????5760
ctttgacgcg?gacgtgcagg?tttcgcacgg?ggggacgatg?gcagcctgag?ccaattccca????5820
gatccccgag?gaatcggcgt?gagcggtcgc?aaaccatccg?gcccggtaca?aatcggcgcg????5880
gcgctgggtg?atgacctggt?ggagaagttg?aaggccgcgc?aggccgccca?gcggcaacgc????5940
atcgaggcag?aagcacgccc?cggtgaatcg?tggcaagcgg?ccgctgatcg?aatccgcaaa????6000
gaatcccggc?aaccgccggc?agccggtgcg?ccgtcgatta?ggaagccgcc?caagggcgac????6060
gagcaaccag?attttttcgt?tccgatgctc?tatgacgtgg?gcacccgcga?tagtcgcagc????6120
atcatggacg?tggccgtttt?ccgtctgtcg?aagcgtgacc?gacgagctgg?cgaggtgatc????6180
cgctacgagc?ttccagacgg?gcacgtagag?gtttccgcag?ggccggccgg?catggccagt????6240
gtgtgggatt?acgacctggt?actgatggcg?gtttcccatc?taaccgaatc?catgaaccga????6300
taccgggaag?ggaagggaga?caagcccggc?cgcgtgttcc?gtccacacgt?tgcggacgta????6360
ctcaagttct?gccggcgagc?cgatggcgga?aagcagaaag?acgacctggt?agaaacctgc????6420
attcggttaa?acaccacgca?cgttgccatg?cagcgtacga?agaaggccaa?gaacggccgc????6480
ctggtgacgg?tatccgaggg?tgaagccttg?attagccgct?acaagatcgt?aaagagcgaa????6540
accgggcggc?cggagtacat?cgagatcgag?ctagctgatt?ggatgtaccg?cgagatcaca????6600
gaaggcaaga?acccggacgt?gctgacggtt?caccccgatt?actttttgat?cgatcccggc????6660
atcggccgtt?ttctctaccg?cctggcacgc?cgcgccgcag?gcaaggcaga?agccagatgg????6720
ttgttcaaga?cgatctacga?acgcagtggc?agcgccggag?agttcaagaa?gttctgtttc????6780
accgtgcgca?agctgatcgg?gtcaaatgac?ctgccggagt?acgatttgaa?ggaggaggcg????6840
gggcaggctg?gcccgatcct?agtcatgcgc?taccgcaacc?tgatcgaggg?cgaagcatcc????6900
gccggttcct?aatgtacgga?gcagatgcta?gggcaaattg?ccctagcagg?ggaaaaaggt????6960
cgaaaaggtc?tctttcctgt?ggatagcacg?tacattggga?acccaaagcc?gtacattggg????7020
aaccggaacc?cgtacattgg?gaacccaaag?ccgtacattg?ggaaccggtc?acacatgtaa????7080
gtgactgata?taaaagagaa?aaaaggcgat?ttttccgcct?aaaactcttt?aaaacttatt????7140
aaaactctta?aaacccgcct?ggcctgtgca?taactgtctg?gccagcgcac?agccgaagag????7200
ctgcaaaaag?cgcctaccct?tcggtcgctg?cgctccctac?gccccgccgc?ttcgcgtcgg????7260
cctatcgcgg?ccgctggccg?ctcaaaaatg?gctggcctac?ggccaggcaa?tctaccaggg????7320
cgcggacaag?ccgcgccgtc?gccactcgac?cgccggcgct?gaggtctgcc?tcgtgaagaa????7380
ggtgttgctg?actcatacca?ggcctgaatc?gccccatcat?ccagccagaa?agtgagggag????7440
ccacggttga?tgagagcttt?gttgtaggtg?gaccagttgg?tgattttgaa?cttttgcttt????7500
gccacggaac?ggtctgcgtt?gtcgggaaga?tgcgtgatct?gatccttcaa?ctcagcaaaa????7560
gttcgattta?ttcaacaaag?ccgccgtccc?gtcaagtcag?cgtaatgctc?tgccagtgtt????7620
acaaccaatt?aaccaattct?gattagaaaa?actcatcgag?catcaaatga?aactgcaatt????7680
tattcatatc?aggattatca?ataccatatt?tttgaaaaag?ccgtttctgt?aatgaaggag????7740
aaaactcacc?gaggcagttc?cataggatgg?caagatcctg?gtatcggtct?gcgattccga????7800
ctcgtccaac?atcaatacaa?cctattaatt?tcccctcgtc?aaaaataagg?ttatcaagtg????7860
agaaatcacc?atgagtgacg?actgaatccg?gtgagaatgg?caaaagctct?gcattaatga????7920
atcggccaac?gcgcggggag?aggcggtttg?cgtattgggc?gctcttccgc?ttcctcgctc????7980
actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca?ctcaaaggcg????8040
gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg?agcaaaaggc????8100
cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc????8160
ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga????8220
ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc????8280
ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcat????8340
agctcacgct?gtaggtatct?cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg????8400
cacgaacccc?ccgttcagcc?cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc????8460
aacccggtaa?gacacgactt?atcgccactg?gcagcagcca?ctggtaacag?gattagcaga????8520
gcgaggtatg?taggcggtgc?tacagagttc?ttgaagtggt?ggcctaacta?cggctacact????8580
agaagaacag?tatttggtat?ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt????8640
ggtagctctt?gatccggcaa?acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag????8700
cagcagatta?cgcgcagaaa?aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg????8760
tctgacgctc?agtggaacga?aaactcacgt?taagggattt?tggtcatgag?attatcaaaa????8820
aggatcttca?cctagatcct?tttgatccgg?aattaattcc?tgtggttggc?atgcacatac????8880
aaatggacga?acggataaac?cttttcacgc?ccttttaaat?atccgattat?tctaataaac????8940
gctcttttct?cttaggttta?cccgccaata?tatcctgtca?aacactgata?gtttaaactg????9000
aaggcgggaa?acgacaatct?gatcatgagc?ggagaattaa?gggagtcacg?ttatgacccc????9060
cgccgatgac?gcgggacaag?ccgttttacg?tttggaactg?acagaaccgc?aacgctgcag????9120
gaattggccg?cagcggccat?ttaaatcaat?tgggcgcgta?cgtagcacta?gtgcgcgatc????9180
gcttaattaa?gcggcgcgcc?taaagcttct?ggcagacaaa?gtggcagaca?tactgtccca????9240
caaatgaaga?tggaatctgt?aaaagaaaac?gcgtgaaata?atgcgtctga?caaaggttag????9300
gtcggctgcc?tttaatcaat?accaaagtgg?tccctaccac?gatggaaaaa?ctgtgcagtc????9360
ggtttggctt?tttctgacga?acaaataaga?ttcgtggccg?acaggtgggg?gtccaccatg????9420
tgaaggcatc?ttcagactcc?aataatggag?caatgacgta?agggcttacg?aaataagtaa????9480
gggtagtttg?ggaaatgtcc?actcacccgt?cagtctataa?atacttagcc?cctccctcat????9540
tgttaaggga?gcaag?????????????????????????????????????????????????????9555
<210>2
<211>9546
<212>DNA
＜213〉artificial
<220>
<223>pNOV2147
<400>2
ggatccccga?tcatgcaaaa?actcattaac?tcagtgcaaa?actatgcctg?gggcagcaaa?????60
acggcgttga?ctgaacttta?tggtatggaa?aatccgtcca?gccagccgat?ggccgagctg????120
tggatgggcg?cacatccgaa?aagcagttca?cgagtgcaga?atgccgccgg?agatatcgtt????180
tcactgcgtg?atgtgattga?gagtgataaa?tcgactctgc?tcggagaggc?cgttgccaaa????240
cgctttggcg?aactgccttt?cctgttcaaa?gtattatgcg?cagcacagcc?actctccatt????300
caggttcatc?caaacaaaca?caattctgaa?atcggttttg?ccaaagaaaa?tgccgcaggt????360
atcccgatgg?atgccgccga?gcgtaactat?aaagatccta?accacaagcc?ggagctggtt????420
tttgcgctga?cgcctttcct?tgcgatgaac?gcgtttcgtg?aattttccga?gattgtctcc????480
ctactccagc?cggtcgcagg?tgcacatccg?gcgattgctc?actttttaca?acagcctgat????540
gccgaacgtt?taagcgaact?gttcgccagc?ctgttgaata?tgcagggtga?agaaaaatcc????600
cgcgcgctgg?cgattttaaa?atcggccctc?gatagccagc?agggtgaacc?gtggcaaacg????660
attcgtttaa?tttctgaatt?ttacccggaa?gacagcggtc?tgttctcccc?gctattgctg????720
aatgtggtga?aattgaaccc?tggcgaagcg?atgttcctgt?tcgctgaaac?accgcacgct????780
tacctgcaag?gcgtggcgct?ggaagtgatg?gcaaactccg?ataacgtgct?gcgtgcgggt????840
ctgacgccta?aatacattga?tattccggaa?ctggttgcca?atgtgaaatt?cgaagccaaa?????900
ccggctaacc?agttgttgac?ccagccggtg?aaacaaggtg?cagaactgga?cttcccgatt?????960
ccagtggatg?attttgcctt?ctcgctgcat?gaccttagtg?ataaagaaac?caccattagc????1020
cagcagagtg?ccgccatttt?gttctgcgtc?gaaggcgatg?caacgttgtg?gaaaggttct????1080
cagcagttac?agcttaaacc?gggtgaatca?gcgtttattg?ccgccaacga?atcaccggtg????1140
actgtcaaag?gccacggccg?tttagcgcgt?gtttacaaca?agctgtaaga?gcttactgaa????1200
aaaattaaca?tctcttgcta?agctgggagc?tctagatccc?cgaatttccc?cgatcgttca????1260
aacatttggc?aataaagttt?cttaagattg?aatcctgttg?ccggtcttgc?gatgattatc????1320
atataatttc?tgttgaatta?cgttaagcat?gtaataatta?acatgtaatg?catgacgtta????1380
tttatgagat?gggtttttat?gattagagtc?ccgcaattat?acatttaata?cgcgatagaa????1440
aacaaaatat?agcgcgcaaa?ctaggataaa?ttatcgcgcg?cggtgtcatc?tatgttacta????1500
gatcgggaat?tgggtaccat?gcccgggcgg?ccagcatggc?cgtatccgca?atgtgttatt????1560
aagttgtcta?agcgtcaatt?tgtttacacc?acaatatatc?ctgccaccag?ccagccaaca????1620
gctccccgac?cggcagctcg?gcacaaaatc?accactcgat?acaggcagcc?catcagaatt????1680
aattctcatg?tttgacagct?tatcatcgac?tgcacggtgc?accaatgctt?ctggcgtcag????1740
gcagccatcg?gaagctgtgg?tatggctgtg?caggtcgtaa?atcactgcat?aattcgtgtc????1800
gctcaaggcg?cactcccgtt?ctggataatg?ttttttgcgc?cgacatcata?acggttctgg????1860
caaatattct?gaaatgagct?gttgacaatt?aatcatccgg?ctcgtataat?gtgtggaatt????1920
gtgagcggat?aacaatttca?cacaggaaac?agaccatgag?ggaagcgttg?atcgccgaag????1980
tatcgactca?actatcagag?gtagttggcg?tcatcgagcg?ccatctcgaa?ccgacgttgc????2040
tggccgtaca?tttgtacggc?tccgcagtgg?atggcggcct?gaagccacac?agtgatattg????2100
atttgctggt?tacggtgacc?gtaaggcttg?atgaaacaac?gcggcgagct?ttgatcaacg????2160
accttttgga?aacttcggct?tcccctggag?agagcgagat?tctccgcgct?gtagaagtca????2220
ccattgttgt?gcacgacgac?atcattccgt?ggcgttatcc?agctaagcgc?gaactgcaat????2280
ttggagaatg?gcagcgcaat?gacattcttg?caggtatctt?cgagccagcc?acgatcgaca????2340
ttgatctggc?tatcttgctg?acaaaagcaa?gagaacatag?cgttgccttg?gtaggtccag????2400
cggcggagga?actctttgat?ccggttcctg?aacaggatct?atttgaggcg?ctaaatgaaa????2460
ccttaacgct?atggaactcg?ccgcccgact?gggctggcga?tgagcgaaat?gtagtgctta????2520
cgttgtcccg?catttggtac?agcgcagtaa?ccggcaaaat?cgcgccgaag?gatgtcgctg????2580
ccgactgggc?aatggagcgc?ctgccggccc?agtatcagcc?cgtcatactt?gaagctaggc????2640
aggcttatct?tggacaagaa?gatcgcttgg?cctcgcgcgc?agatcagttg?gaagaatttg????2700
ttcactacgt?gaaaggcgag?atcaccaaag?tagtcggcaa?ataaagctct?agtggatctc????2760
cgtacccccg?ggggatctgg?ctcgcggcgg?acgcacgacg?ccggggcgag?accataggcg????2820
atctcctaaa?tcaatagtag?ctgtaacctc?gaagcgtttc?acttgtaaca?acgattgaga????2880
atttttgtca?taaaattgaa?atacttggtt?cgcatttttg?tcatccgcgg?tcagccgcaa????2940
ttctgacgaa?ctgcccattt?agctggagat?gattgtacat?ccttcacgtg?aaaatttctc????3000
aagcgctgtg?aacaagggtt?cagattttag?attgaaaggt?gagccgttga?aacacgttct????3060
tcttgtcgat?gacgacgtcg?ctatgcggca?tcttattatt?gaatacctta?cgatccacgc????3120
cttcaaagtg?accgcggtag?ccgacagcac?ccagttcaca?agagtactct?cttccgcgac????3180
ggtcgatgtc?gtggttgttg?atctaaattt?aggtcgtgaa?gatgggctcg?agatcgttcg????3240
taatctggcg?gcaaagtctg?atattccaat?cataattatc?agtggcgacc?gccttgagga????3300
gacggataaa?gttgttgcac?tcgagctagg?agcaagtgat?tttatcgcta?agccgttcag????3360
tatcagagag?tttctagcac?gcattcgggt?tgccttgcgc?gtgcgcccca?acgttgtccg????3420
ctccaaagac?cgacggtctt?tttgttttac?tgactggaca?cttaatctca?ggcaacgtcg????3480
cttgatgtcc?gaagctggcg?gtgaggtgaa?acttacggca?ggtgagttca?atcttctcct????3540
cgcgttttta?gagaaacccc?gcgacgttct?atcgcgcgag?caacttctca?ttgccagtcg????3600
agtacgcgac?gaggaggttt?atgacaggag?tatagatgtt?ctcattttga?ggctgcgccg????3660
caaacttgag?gcagatccgt?caagccctca?actgataaaa?acagcaagag?gtgccggtta????3720
tttctttgac?gcggacgtgc?aggtttcgca?cggggggacg?atggcagcct?gagccaattc????3780
ccagatcccc?gaggaatcgg?cgtgagcggt?cgcaaaccat?ccggcccggt?acaaatcggc????3840
gcggcgctgg?gtgatgacct?ggtggagaag?ttgaaggccg?cgcaggccgc?ccagcggcaa????3900
cgcatcgagg?cagaagcacg?ccccggtgaa?tcgtggcaag?cggccgctga?tcgaatccgc????3960
aaagaatccc?ggcaaccgcc?ggcagccggt?gcgccgtcga?ttaggaagcc?gcccaagggc????4020
gacgagcaac?cagatttttt?cgttccgatg?ctctatgacg?tgggcacccg?cgatagtcgc????4080
agcatcatgg?acgtggccgt?tttccgtctg?tcgaagcgtg?accgacgagc?tggcgaggtg????4140
atccgctacg?agcttccaga?cgggcacgta?gaggtttccg?cagggccggc?cggcatggcc????4200
agtgtgtggg?attacgacct?ggtactgatg?gcggtttccc?atctaaccga?atccatgaac????4260
cgataccggg?aagggaaggg?agacaagccc?ggccgcgtgt?tccgtccaca?cgttgcggac????4320
gtactcaagt?tctgccggcg?agccgatggc?ggaaagcaga?aagacgacct?ggtagaaacc????4380
tgcattcggt?taaacaccac?gcacgttgcc?atgcagcgta?cgaagaaggc?caagaacggc????4440
cgcctggtga?cggtatccga?gggtgaagcc?ttgattagcc?gctacaagat?cgtaaagagc????4500
gaaaccgggc?ggccggagta?catcgagatc?gagctagctg?attggatgta?ccgcgagatc????4560
acagaaggca?agaacccgga?cgtgctgacg?gttcaccccg?attacttttt?gatcgatccc????4620
ggcatcggcc?gttttctcta?ccgcctggca?cgccgcgccg?caggcaaggc?agaagccaga????4680
tggttgttca?agacgatcta?cgaacgcagt?ggcagcgccg?gagagttcaa?gaagttctgt????4740
ttcaccgtgc?gcaagctgat?cgggtcaaat?gacctgccgg?agtacgattt?gaaggaggag????4800
gcggggcagg?ctggcccgat?cctagtcatg?cgctaccgca?acctgatcga?gggcgaagca????4860
tccgccggtt?cctaatgtac?ggagcagatg?ctagggcaaa?ttgccctagc?aggggaaaaa????4920
ggtcgaaaag?gtctctttcc?tgtggatagc?acgtacattg?ggaacccaaa?gccgtacatt????4980
gggaaccgga?acccgtacat?tgggaaccca?aagccgtaca?ttgggaaccg?gtcacacatg????5040
taagtgactg?atataaaaga?gaaaaaaggc?gatttttccg?cctaaaactc?tttaaaactt????5100
attaaaactc?ttaaaacccg?cctggcctgt?gcataactgt?ctggccagcg?cacagccgaa????5160
gagctgcaaa?aagcgcctac?ccttcggtcg?ctgcgctccc?tacgccccgc?cgcttcgcgt????5220
cggcctatcg?cggccgctgg?ccgctcaaaa?atggctggcc?tacggccagg?caatctacca????5280
gggcgcggac?aagccgcgcc?gtcgccactc?gaccgccggc?gctgaggtct?gcctcgtgaa????5340
gaaggtgttg?ctgactcata?ccaggcctga?atcgccccat?catccagcca?gaaagtgagg????5400
gagccacggt?tgatgagagc?tttgttgtag?gtggaccagt?tggtgatttt?gaacttttgc????5460
tttgccacgg?aacggtctgc?gttgtcggga?agatgcgtga?tctgatcctt?caactcagca????5520
aaagttcgat?ttattcaaca?aagccgccgt?cccgtcaagt?cagcgtaatg?ctctgccagt????5580
gttacaacca?attaaccaat?tctgattaga?aaaactcatc?gagcatcaaa?tgaaactgca????5640
atttattcat?atcaggatta?tcaataccat?atttttgaaa?aagccgtttc?tgtaatgaag????5700
gagaaaactc?accgaggcag?ttccatagga?tggcaagatc?ctggtatcgg?tctgcgattc????5760
cgactcgtcc?aacatcaata?caacctatta?atttcccctc?gtcaaaaata?aggttatcaa????5820
gtgagaaatc?accatgagtg?acgactgaat?ccggtgagaa?tggcaaaagc?tctgcattaa????5880
tgaatcggcc?aacgcgcggg?gagaggcggt?ttgcgtattg?ggcgctcttc?cgcttcctcg????5940
ctcactgact?cgctgcgctc?ggtcgttcgg?ctgcggcgag?cggtatcagc?tcactcaaag????6000
gcggtaatac?ggttatccac?agaatcaggg?gataacgcag?gaaagaacat?gtgagcaaaa????6060
ggccagcaaa?aggccaggaa?ccgtaaaaag?gccgcgttgc?tggcgttttt?ccataggctc????6120
cgcccccctg?acgagcatca?caaaaatcga?cgctcaagtc?agaggtggcg?aaacccgaca????6180
ggactataaa?gataccaggc?gtttccccct?ggaagctccc?tcgtgcgctc?tcctgttccg????6240
accctgccgc?ttaccggata?cctgtccgcc?tttctccctt?cgggaagcgt?ggcgctttct????6300
catagctcac?gctgtaggta?tctcagttcg?gtgtaggtcg?ttcgctccaa?gctgggctgt????6360
gtgcacgaac?cccccgttca?gcccgaccgc?tgcgccttat?ccggtaacta?tcgtcttgag????6420
tccaacccgg?taagacacga?cttatcgcca?ctggcagcag?ccactggtaa?caggattagc????6480
agagcgaggt?atgtaggcgg?tgctacagag?ttcttgaagt?ggtggcctaa?ctacggctac????6540
actagaagaa?cagtatttgg?tatctgcgct?ctgctgaagc?cagttacctt?cggaaaaaga????6600
gttggtagct?cttgatccgg?caaacaaacc?accgctggta?gcggtggttt?ttttgtttgc????6660
aagcagcaga?ttacgcgcag?aaaaaaagga?tctcaagaag?atcctttgat?cttttctacg????6720
gggtctgacg?ctcagtggaa?cgaaaactca?cgttaaggga?ttttggtcat?gagattatca????6780
aaaaggatct?tcacctagat?ccttttgatc?cggaattaat?tcctgtggtt?ggcatgcaca????6840
tacaaatgga?cgaacggata?aaccttttca?cgccctttta?aatatccgat?tattctaata????6900
aacgctcttt?tctcttaggt?ttacccgcca?atatatcctg?tcaaacactg?atagtttaaa????6960
ctgaaggcgg?gaaacgacaa?tctgatcatg?agcggagaat?taagggagtc?acgttatgac????7020
ccccgccgat?gacgcgggac?aagccgtttt?acgtttggaa?ctgacagaac?cgcaacgctg????7080
caggaattgg?ccgcagcggc?catttaaatc?aattgggcgc?gtacgtagca?ctagtgcgcg????7140
atcgcttaat?taagcggcgc?gcctaaagct?tctggcagac?aaagtggcag?acatactgtc????7200
ccacaaatga?agatggaatc?tgtaaaagaa?aacgcgtgaa?ataatgcgtc?tgacaaaggt????7260
taggtcggct?gcctttaatc?aataccaaag?tggtccctac?cacgatggaa?aaactgtgca????7320
gtcggtttgg?ctttttctga?cgaacaaata?agattcgtgg?ccgacaggtg?ggggtccacc????7380
atgtgaaggc?atcttcagac?tccaataatg?gagcaatgac?gtaagggctt?acgaaataag????7440
taagggtagt?ttgggaaatg?tccactcacc?cgtcagtcta?taaatactta?gcccctccct????7500
cattgttaag?ggagcaagga?tccaccggtc?gccaccatgg?ccctgtccaa?caagttcatc????7560
ggcgacgaca?tgaagatgac?ctaccacatg?gacggctgcg?tgaacggcca?ctacttcacc????7620
gtgaagggcg?agggcagcgg?caagccctac?gagggcaccc?agacctccac?cttcaaggtg????7680
accatggcca?acggcggccc?cctggccttc?tccttcgaca?tcctgtccac?cgtgttcatg????7740
tacggcaacc?gctgcttcac?cgcctacccc?accagcatgc?ccgactactt?caagcaggcc????7800
ttccccgacg?gcatgtccta?cgagagaacc?ttcacctacg?aggacggcgg?cgtggccacc????7860
gccagctggg?agatcagcct?gaagggcaac?tgcttcgagc?acaagtccac?cttccacggc????7920
gtgaacttcc?ccgccgacgg?ccccgtgatg?gccaagaaga?ccaccggctg?ggacccctcc????7980
ttcgagaaga?tgaccgtgtg?cgacggcatc?ttgaagggcg?acgtgaccgc?cttcctgatg????8040
ctgcagggcg?gcggcaacta?cagatgccag?ttccacacct?cctacaagac?caagaagccc????8100
gtgaccatgc?cccccaacca?cgtggtggag?caccgcatcg?ccagaaccga?cctggacaag????8160
ggcggcaaca?gcgtgcagct?gaccgagcac?gccgtggccc?acatcacctc?cgtggtgccc????8220
ttctgagagc?tctagatccc?cgaatttccc?cgatcgttca?aacatttggc?aataaagttt????8280
cttaagattg?aatcctgttg?ccggtcttgc?gatgattatc?atataatttc?tgttgaatta????8340
cgttaagcat?gtaataatta?acatgtaatg?catgacgtta?tttatgagat?gggtttttat????8400
gattagagtc?ccgcaattat?acatttaata?cgcgatagaa?aacaaaatat?agcgcgcaaa????8460
ctaggataaa?ttatcgcgcg?cggtgtcatc?tatgttacta?gatcgggaat?tgggtaccga????8520
attcactggc?cgtcgtttta?caacgtcgtg?actgggaaaa?ccctggcgtt?acccaactta????8580
atcgccttgc?agcacatccc?cctttcgcca?gctggcgtaa?tagcgaagag?gcccgcaccg????8640
atcgcccttc?ccaacagttg?cgcagcctga?atggcgaatg?gcgcctgatg?cggtattttc????8700
tccttacgca?tctgtgcggt?atttcacacc?gcatatggtg?cactctcagt?acaatctgct????8760
ctgatgccgc?atagttaagc?cagccccgac?acccgccaac?acccgctgac?gcgccctgac????8820
gggcttgtct?gctcccggca?tccgcttaca?gacaagctgt?gaccgtctcc?gggagctgca????8880
tgtgtcagag?gttttcaccg?tcatcaccga?aacgcgcgag?acgaaagggc?ctcgtgatac????8940
gcctattttt?ataggttaat?gtcatgataa?taatggtttc?ttagacgtca?ggtggcactt????9000
ttcggggaaa?tgtgcgcgga?acccctattt?gtttattttt?ctaaatacat?tcaaatatgt????9060
atccgctcat?gagacaataa?ccctgataaa?tgcttcaatg?gcgcgccggt?accagcttgc????9120
atgcctgcag?gtcgactcta?gaggatcctg?gcagacaaag?tggcagacat?actgtcccac????9180
aaatgaagat?ggaatctgta?aaagaaaacg?cgtgaaataa?tgcgtctgac?aaaggttagg????9240
tcggctgcct?ttaatcaata?ccaaagtggt?ccctaccacg?atggaaaaac?tgtgcagtcg????9300
gtttggcttt?ttctgacgaa?caaataagat?tcgtggccga?caggtggggg?tccaccatgt????9360
gaaggcatct?tcagactcca?ataatggagc?aatgacgtaa?gggcttacga?aataagtaag????9420
ggtagtttgg?gaaatgtcca?ctcacccgtc?agtctataaa?tacttagccc?ctccctcatt????9480
gttaagggag?caaaatctca?gagagatagt?cctagagaga?gaaagagagc?aagtagccta????9540
gaagta???????????????????????????????????????????????????????????????9546
<210>3
<211>10604
<212>DNA
＜213〉artificial
<220>
<223>pBSC11234
<400>3
cgcgcctaaa?gcttgcatgc?ctgcaggtcg?actctagagg?atcctggcag?acaaagtggc?????60
agacatactg?tcccacaaat?gaagatggaa?tctgtaaaag?aaaacgcgtg?aaataatgcg????120
tctgacaaag?gttaggtcgg?ctgcctttaa?tcaataccaa?agtggtccct?accacgatgg????180
aaaaactgtg?cagtcggttt?ggctttttct?gacgaacaaa?taagattcgt?ggccgacagg????240
tgggggtcca?ccatgtgaag?gcatcttcag?actccaataa?tggagcaatg?acgtaagggc????300
ttacgaaata?agtaagggta?gtttgggaaa?tgtccactca?cccgtcagtc?tataaatact????360
tagcccctcc?ctcattgtta?agggagcaaa?atctcagaga?gatagtccta?gagagagaaa?????420
gagagcaagt?agcctagaag?taggatcccc?gatcatgcaa?aaactcatta?actcagtgca?????480
aaactatgcc?tggggcagca?aaacggcgtt?gactgaactt?tatggtatgg?aaaatccgtc?????540
cagccagccg?atggccgagc?tgtggatggg?cgcacatccg?aaaagcagtt?cacgagtgca?????600
gaatgccgcc?ggagatatcg?tttcactgcg?tgatgtgatt?gagagtgata?aatcgactct?????660
gctcggagag?gccgttgcca?aacgctttgg?cgaactgcct?ttcctgttca?aagtattatg?????720
cgcagcacag?ccactctcca?ttcaggttca?tccaaacaaa?cacaattctg?aaatcggttt?????780
tgccaaagaa?aatgccgcag?gtatcccgat?ggatgccgcc?gagcgtaact?ataaagatcc?????840
taaccacaag?ccggagctgg?tttttgcgct?gacgcctttc?cttgcgatga?acgcgtttcg?????900
tgaattttcc?gagattgtct?ccctactcca?gccggtcgca?ggtgcacatc?cggcgattgc?????960
tcacttttta?caacagcctg?atgccgaacg?tttaagcgaa?ctgttcgcca?gcctgttgaa????1020
tatgcagggt?gaagaaaaat?cccgcgcgct?ggcgatttta?aaatcggccc?tcgatagcca????1080
gcagggtgaa?ccgtggcaaa?cgattcgttt?aatttctgaa?ttttacccgg?aagacagcgg????1140
tctgttctcc?ccgctattgc?tgaatgtggt?gaaattgaac?cctggcgaag?cgatgttcct????1200
gttcgctgaa?acaccgcacg?cttacctgca?aggcgtggcg?ctggaagtga?tggcaaactc????1260
cgataacgtg?ctgcgtgcgg?gtctgacgcc?taaatacatt?gatattccgg?aactggttgc????1320
caatgtgaaa?ttcgaagcca?aaccggctaa?ccagttgttg?acccagccgg?tgaaacaagg????1380
tgcagaactg?gacttcccga?ttccagtgga?tgattttgcc?ttctcgctgc?atgaccttag????1440
tgataaagaa?accaccatta?gccagcagag?tgccgccatt?ttgttctgcg?tcgaaggcga????1500
tgcaacgttg?tggaaaggtt?ctcagcagtt?acagcttaaa?ccgggtgaat?cagcgtttat????1560
tgccgccaac?gaatcaccgg?tgactgtcaa?aggccacggc?cgtttagcgc?gtgtttacaa????1620
caagctgtaa?gagcttactg?aaaaaattaa?catctcttgc?taagctggga?gctctagatc????1680
cccgaatttc?cccgatcgtt?caaacatttg?gcaataaagt?ttcttaagat?tgaatcctgt????1740
tgccggtctt?gcgatgatta?tcatataatt?tctgttgaat?tacgttaagc?atgtaataat????1800
taacatgtaa?tgcatgacgt?tatttatgag?atgggttttt?atgattagag?tcccgcaatt????1860
atacatttaa?tacgcgatag?aaaacaaaat?atagcgcgca?aactaggata?aattatcgcg????1920
cgcggtgtca?tctatgttac?tagatcggga?attgggtacc?atgcccgggc?ggccagcatg????1980
gccgtatccg?caatgtgtta?ttaagttgtc?taagcgtcaa?tttgtttaca?ccacaatata????2040
tcctgccacc?agccagccaa?cagctccccg?accggcagct?cggcacaaaa?tcaccactcg????2100
atacaggcag?cccatcagaa?ttaattctca?tgtttgacag?cttatcatcg?actgcacggt????2160
gcaccaatgc?ttctggcgtc?aggcagccat?cggaagctgt?ggtatggctg?tgcaggtcgt????2220
aaatcactgc?ataattcgtg?tcgctcaagg?cgcactcccg?ttctggataa?tgttttttgc????2280
gccgacatca?taacggttct?ggcaaatatt?ctgaaatgag?ctgttgacaa?ttaatcatcc????2340
ggctcgtata?atgtgtggaa?ttgtgagcgg?ataacaattt?cacacaggaa?acagaccatg????2400
agggaagcgt?tgatcgccga?agtatcgact?caactatcag?aggtagttgg?cgtcatcgag????2460
cgccatctcg?aaccgacgtt?gctggccgta?catttgtacg?gctccgcagt?ggatggcggc????2520
ctgaagccac?acagtgatat?tgatttgctg?gttacggtga?ccgtaaggct?tgatgaaaca????2580
acgcggcgag?ctttgatcaa?cgaccttttg?gaaacttcgg?cttcccctgg?agagagcgag????2640
attctccgcg?ctgtagaagt?caccattgtt?gtgcacgacg?acatcattcc?gtggcgttat????2700
ccagctaagc?gcgaactgca?atttggagaa?tggcagcgca?atgacattct?tgcaggtatc????2760
ttcgagccag?ccacgatcga?cattgatctg?gctatcttgc?tgacaaaagc?aagagaacat????2820
agcgttgcct?tggtaggtcc?agcggcggag?gaactctttg?atccggttcc?tgaacaggat????2880
ctatttgagg?cgctaaatga?aaccttaacg?ctatggaact?cgccgcccga?ctgggctggc????2940
gatgagcgaa?atgtagtgct?tacgttgtcc?cgcatttggt?acagcgcagt?aaccggcaaa????3000
atcgcgccga?aggatgtcgc?tgccgactgg?gcaatggagc?gcctgccggc?ccagtatcag????3060
cccgtcatac?ttgaagctag?gcaggcttat?cttggacaag?aagatcgctt?ggcctcgcgc????3120
gcagatcagt?tggaagaatt?tgttcactac?gtgaaaggcg?agatcaccaa?agtagtcggc????3180
aaataaagct?ctagtggatc?tccgtacccc?cgggggatct?ggctcgcggc?ggacgcacga????3240
cgccggggcg?agaccatagg?cgatctccta?aatcaatagt?agctgtaacc?tcgaagcgtt????3300
tcacttgtaa?caacgattga?gaatttttgt?cataaaattg?aaatacttgg?ttcgcatttt????3360
tgtcatccgc?ggtcagccgc?aattctgacg?aactgcccat?ttagctggag?atgattgtac????3420
atccttcacg?tgaaaatttc?tcaagcgctg?tgaacaaggg?ttcagatttt?agattgaaag????3480
gtgagccgtt?gaaacacgtt?cttcttgtcg?atgacgacgt?cgctatgcgg?catcttatta????3540
ttgaatacct?tacgatccac?gccttcaaag?tgaccgcggt?agccgacagc?acccagttca????3600
caagagtact?ctcttccgcg?acggtcgatg?tcgtggttgt?tgatctaaat?ttaggtcgtg????3660
aagatgggct?cgagatcgtt?cgtaatctgg?cggcaaagtc?tgatattcca?atcataatta????3720
tcagtggcga?ccgccttgag?gagacggata?aagttgttgc?actcgagcta?ggagcaagtg????3780
attttatcgc?taagccgttc?agtatcagag?agtttctagc?acgcattcgg?gttgccttgc????3840
gcgtgcgccc?caacgttgtc?cgctccaaag?accgacggtc?tttttgtttt?actgactgga????3900
cacttaatct?caggcaacgt?cgcttgatgt?ccgaagctgg?cggtgaggtg?aaacttacgg????3960
caggtgagtt?caatcttctc?ctcgcgtttt?tagagaaacc?ccgcgacgtt?ctatcgcgcg????4020
agcaacttct?cattgccagt?cgagtacgcg?acgaggaggt?ttatgacagg?agtatagatg????4080
ttctcatttt?gaggctgcgc?cgcaaacttg?aggcagatcc?gtcaagccct?caactgataa????4140
aaacagcaag?aggtgccggt?tatttctttg?acgcggacgt?gcaggtttcg?cacgggggga????4200
cgatggcagc?ctgagccaat?tcccagatcc?ccgaggaatc?ggcgtgagcg?gtcgcaaacc????4260
atccggcccg?gtacaaatcg?gcgcggcgct?gggtgatgac?ctggtggaga?agttgaaggc????4320
cgcgcaggcc?gcccagcggc?aacgcatcga?ggcagaagca?cgccccggtg?aatcgtggca????4380
agcggccgct?gatcgaatcc?gcaaagaatc?ccggcaaccg?ccggcagccg?gtgcgccgtc????4440
gattaggaag?ccgcccaagg?gcgacgagca?accagatttt?ttcgttccga?tgctctatga????4500
cgtgggcacc?cgcgatagtc?gcagcatcat?ggacgtggcc?gttttccgtc?tgtcgaagcg????4560
tgaccgacga?gctggcgagg?tgatccgcta?cgagcttcca?gacgggcacg?tagaggtttc????4620
cgcagggccg?gccggcatgg?ccagtgtgtg?ggattacgac?ctggtactga?tggcggtttc????4680
ccatctaacc?gaatccatga?accgataccg?ggaagggaag?ggagacaagc?ccggccgcgt????4740
gttccgtcca?cacgttgcgg?acgtactcaa?gttctgccgg?cgagccgatg?gcggaaagca????4800
gaaagacgac?ctggtagaaa?cctgcattcg?gttaaacacc?acgcacgttg?ccatgcagcg????4860
tacgaagaag?gccaagaacg?gccgcctggt?gacggtatcc?gagggtgaag?ccttgattag????4920
ccgctacaag?atcgtaaaga?gcgaaaccgg?gcggccggag?tacatcgaga?tcgagctagc????4980
tgattggatg?taccgcgaga?tcacagaagg?caagaacccg?gacgtgctga?cggttcaccc????5040
cgattacttt?ttgatcgatc?ccggcatcgg?ccgttttctc?taccgcctgg?cacgccgcgc????5100
cgcaggcaag?gcagaagcca?gatggttgtt?caagacgatc?tacgaacgca?gtggcagcgc????5160
cggagagttc?aagaagttct?gtttcaccgt?gcgcaagctg?atcgggtcaa?atgacctgcc????5220
ggagtacgat?ttgaaggagg?aggcggggca?ggctggcccg?atcctagtca?tgcgctaccg????5280
caacctgatc?gagggcgaag?catccgccgg?ttcctaatgt?acggagcaga?tgctagggca????5340
aattgcccta?gcaggggaaa?aaggtcgaaa?aggtctcttt?cctgtggata?gcacgtacat????5400
tgggaaccca?aagccgtaca?ttgggaaccg?gaacccgtac?attgggaacc?caaagccgta????5460
cattgggaac?cggtcacaca?tgtaagtgac?tgatataaaa?gagaaaaaag?gcgatttttc????5520
cgcctaaaac?tctttaaaac?ttattaaaac?tcttaaaacc?cgcctggcct?gtgcataact????5580
gtctggccag?cgcacagccg?aagagctgca?aaaagcgcct?acccttcggt?cgctgcgctc????5640
cctacgcccc?gccgcttcgc?gtcggcctat?cgcggccgct?ggccgctcaa?aaatggctgg????5700
cctacggcca?ggcaatctac?cagggcgcgg?acaagccgcg?ccgtcgccac?tcgaccgccg????5760
gcgctgaggt?ctgcctcgtg?aagaaggtgt?tgctgactca?taccaggcct?gaatcgcccc????5820
atcatccagc?cagaaagtga?gggagccacg?gttgatgaga?gctttgttgt?aggtggacca????5880
gttggtgatt?ttgaactttt?gctttgccac?ggaacggtct?gcgttgtcgg?gaagatgcgt????5940
gatctgatcc?ttcaactcag?caaaagttcg?atttattcaa?caaagccgcc?gtcccgtcaa????6000
gtcagcgtaa?tgctctgcca?gtgttacaac?caattaacca?attctgatta?gaaaaactca????6060
tcgagcatca?aatgaaactg?caatttattc?atatcaggat?tatcaatacc?atatttttga????6120
aaaagccgtt?tctgtaatga?aggagaaaac?tcaccgaggc?agttccatag?gatggcaaga????6180
tcctggtatc?ggtctgcgat?tccgactcgt?ccaacatcaa?tacaacctat?taatttcccc????6240
tcgtcaaaaa?taaggttatc?aagtgagaaa?tcaccatgag?tgacgactga?atccggtgag????6300
aatggcaaaa?gctctgcatt?aatgaatcgg?ccaacgcgcg?gggagaggcg?gtttgcgtat????6360
tgggcgctct?tccgcttcct?cgctcactga?ctcgctgcgc?tcggtcgttc?ggctgcggcg????6420
agcggtatca?gctcactcaa?aggcggtaat?acggttatcc?acagaatcag?gggataacgc????6480
aggaaagaac?atgtgagcaa?aaggccagca?aaaggccagg?aaccgtaaaa?aggccgcgtt????6540
gctggcgttt?ttccataggc?tccgcccccc?tgacgagcat?cacaaaaatc?gacgctcaag????6600
tcagaggtgg?cgaaacccga?caggactata?aagataccag?gcgtttcccc?ctggaagctc????6660
cctcgtgcgc?tctcctgttc?cgaccctgcc?gcttaccgga?tacctgtccg?cctttctccc????6720
ttcgggaagc?gtggcgcttt?ctcatagctc?acgctgtagg?tatctcagtt?cggtgtaggt????6780
cgttcgctcc?aagctgggct?gtgtgcacga?accccccgtt?cagcccgacc?gctgcgcctt????6840
atccggtaac?tatcgtcttg?agtccaaccc?ggtaagacac?gacttatcgc?cactggcagc????6900
agccactggt?aacaggatta?gcagagcgag?gtatgtaggc?ggtgctacag?agttcttgaa????6960
gtggtggcct?aactacggct?acactagaag?aacagtattt?ggtatctgcg?ctctgctgaa????7020
gccagttacc?ttcggaaaaa?gagttggtag?ctcttgatcc?ggcaaacaaa?ccaccgctgg????7080
tagcggtggt?ttttttgttt?gcaagcagca?gattacgcgc?agaaaaaaag?gatctcaaga????7140
agatcctttg?atcttttcta?cggggtctga?cgctcagtgg?aacgaaaact?cacgttaagg????7200
gattttggtc?atgagattat?caaaaaggat?cttcacctag?atccttttga?tccggaatta????7260
attcctgtgg?ttggcatgca?catacaaatg?gacgaacgga?taaacctttt?cacgcccttt????7320
taaatatccg?attattctaa?taaacgctct?tttctcttag?gtttacccgc?caatatatcc????7380
tgtcaaacac?tgatagttta?aactgaaggc?gggaaacgac?aatctgatca?tgagcggaga????7440
attaagggag?tcacgttatg?acccccgccg?atgacgcggg?acaagccgtt?ttacgtttgg????7500
aactgacaga?accgcaacgc?tgcaggaatt?ggccgcagcg?gccatttaaa?tcaattgggc????7560
gcgtacgtag?cactagtgcg?cgatcgctta?attaagcggc?gcgcctgcag?gcggccgcac????7620
aattattata?tcaaaatggc?aaaaacattt?aatacgtatt?atttaagaaa?aaaatatgta????7680
ataatatatt?tatattttaa?tatctattct?tatgtatttt?ttaaaaatct?attatatatt????7740
gatcaactaa?aatattttta?tatctacact?tattttgcat?ttttatcaat?tttcttgcgt????7800
tttttggcat?atttaataat?gactattctt?taataatcga?tcattattct?tacatggtac????7860
atattgttgg?aaccatatga?agtgtccatt?gcatttgact?atgtggatag?tgttttgatc????7920
caggcctcca?tttgccgctt?attaattaat?ttggtaacag?tccgtactaa?tcagttactt????7980
atccttcctc?catcataatt?aatcttggta?gtctcgaatg?ccacaacact?gactagtctc????8040
ttggatcata?agaaaaagcc?aaggaacaaa?agaagacaaa?acacaatggg?agtatccttt????8100
gcatagcaat?gtctaagttc?ataaaattca?aacaaaaacg?caatcacaca?cagtggacat????8160
cacttatcca?ctagctgatc?aggatcgccg?cgtcaagaaa?aaaaaactgg?accccaaaag????8220
ccatgcacaa?caacacgtac?tcacaaaggt?gtcaatcgag?cagcccaaaa?cattcaccaa????8280
ctcaacccat?catgagccca?cacatttgtt?gtttctaacc?caacctcaaa?ctcgtattct????8340
cttccgccac?ctcatttttg?tttatttcaa?cacccgtcaa?actgcatgcc?accccgtggc????8400
caaatgtcca?tgcatgttaa?caagacctat?gactataaat?atctgcaatc?tcggcccagg????8460
ttttcatcat?caagaaccag?ttcaatatcc?tagtacaccg?tattaaagaa?tttaagatat????8520
actccaccgg?atccaccatg?gccaagctag?ttttttccct?ttgttttctg?cttttcagtg????8580
gctgctgctt?cgctgagatt?ttcggcaaga?ccttccgcga?gggccgcttc?gtgctcaagg????8640
agaagaactt?caccgtggag?ttcgccgtgg?agaagatcca?cctcggctgg?aagatatcgg????8700
gccgcgtgaa?gggctcgccg?ggccgcctcg?aggtgctccg?caccaaggcc?ccggagaagg????8760
tgctcgtgaa?caactggcag?tcctggggcc?cgtgccgcgt?ggtggacgcc?ttctccttca????8820
agccgccgga?gatcgacccg?aactggcgct?acaccgcatc?cgtggtgccg?gacgtgctcg????8880
agcgcaacct?gcagtccgac?tacttcgtgg?ccgaggaggg?caaggtgtac?ggcttcctct????8940
cctccaagat?cgcccacccg?ttcttcgcgg?tggaggacgg?cgagctggtg?gcctacctcg????9000
agtacttcga?cgtggagttc?gacgacttcg?tgccgctgga?gccgctcgtg?gtgctcgagg????9060
acccgaacac?cccgctcctc?ctcgagaagt?acgccgagct?ggtgggcatg?gagaacaacg????9120
cccgggtgcc?gaagcacacg?ccgaccggct?ggtgctcctg?gtatcactac?ttcctcgacc????9180
tcacctggga?ggagaccctc?aagaacctca?agctcgccaa?gaacttcccg?ttcgaggtgt????9240
tccagatcga?cgacgcctac?gagaaggaca?tcggcgactg?gctcgtgacc?cgcggcgact????9300
tcccgtccgt?ggaggagatg?gccaaggtga?tcgccgagaa?cggcttcatc?cccggcatct????9360
ggaccgcccc?gttctccgtg?tccgagacta?gtgacgtgtt?caacgagcac?ccggactggg????9420
tggtgaagga?gaacggcgag?ccgaagatgg?cctaccgcaa?ctggaacaag?aagatttacg????9480
ccctcgacct?ctccaaggac?gaggtgctca?actggctctt?cgacctcttc?tcctccctcc????9540
gcaagatggg?ctaccgctac?ttcaagatcg?acttcctctt?cgcgggcgcc?gtgccggggg????9600
agcgcaagaa?gaacatcacc?ccgatccagg?ccttccgcaa?gggcatcgag?accatccgca????9660
aggccgtggg?ggaggactcc?ttcatcctcg?gctgcggctc?ccccctcctc?ccggccgtgg????9720
gctgcgtgga?tggcatgcgc?atcggcccgg?acaccgcccc?gttctgggga?gagcacatcg????9780
aggacaacgg?cgccccggcg?gcccgctggg?ccctccgcaa?cgccatcacc?cgctacttca????9840
tgcacgaccg?cttctggctc?aacgacccgg?actgcctcat?cctccgcgag?gagaagaccg???9900
acctcaccca?gaaggagaag?gagctgtact?cctacacctg?cggcgttcta?gacaacatga???9960
tcatcgagtc?cgacgacctc?tccctcgtgc?gcgaccacgg?caagaaggtg?ctcaaggaga??10020
ccctcgagct?gctcgggggc?aggccgcgcg?tgcagaacat?catgtccgag?gacctccgct??10080
acgagatcgt?gtcctcgggc?accctctccg?gcaacgtgaa?gatcgtggtg?gacctcaact??10140
cccgcgagta?ccacctcgag?aaggagggca?agtcctccct?caagaagcgc?gtggtgaagc??10200
gggaggacgg?caggaacttc?tacttctacg?aggagggcga?gcgcgagtga?aagcttgacg??10260
tcactagtgc?gatcgcgcta?gccatggccg?gcctaggcgc?ccgggagatc?cccgaatttc??10320
cccgatcgtt?caaacatttg?gcaataaagt?ttcttaagat?tgaatcctgt?tgccggtctt??10380
gcgatgatta?tcatataatt?tctgttgaat?tacgttaagc?atgtaataat?taacatgtaa??10440
tgcatgacgt?tatttatgag?atgggttttt?atgattagag?tcccgcaatt?atacatttaa??10500
tacgcgatag?aaaacaaaat?atagcgcgca?aactaggata?aattatcgcg?cgcggtgtca??10560
tctatgttac?tagatcggga?attcctcgag?tctagacctg?cagg???????????????????10604
<210>4
<211>8757
<212>DNA
＜213〉artificial
<220>
<223>pBSC11369
<400>4
aagcttctgg?cagacaaagt?ggcagacata?ctgtcccaca?aatgaagatg?gaatctgtaa?????60
aagaaaacgc?gtgaaataat?gcgtctgaca?aaggttaggt?cggctgcctt?taatcaatac????120
caaagtggtc?cctaccacga?tggaaaaact?gtgcagtcgg?tttggctttt?tctgacgaac????180
aaataagatt?cgtggccgac?aggtgggggt?ccaccatgtg?aaggcatctt?cagactccaa????240
taatggagca?atgacgtaag?ggcttacgaa?ataagtaagg?gtagtttggg?aaatgtccac????300
tcacccgtca?gtctataaat?acttagcccc?tccctcattg?ttaagggagc?aaggatccac????360
cggtcgccac?catggcccag?tccaagcacg?gcctgaccaa?ggagatgacc?atgaagtacc????420
gcatggaggg?ctgcgtggac?ggccacaagt?tcgtgatcac?cggcgagggc?atcggctacc????480
ccttcaaggg?caagcaggcc?atcaacctgt?gcgtggtgga?gggcggcccc?ttgcccttcg????540
ccgaggacat?cttgtccgcc?gccttcatgt?acggcaaccg?cgtgttcacc?gagtaccccc????600
aggacatcgt?cgactacttc?aagaactcct?gccccgccgg?ctacacctgg?gaccgctcct????660
tcctgttcga?ggacggcgcc?gtgtgcatct?gcaacgccga?catcaccgtg?agcgtggagg????720
agaactgcat?gtaccacgag?tccaagttct?acggcgtgaa?cttccccgcc?gacggccccg????780
tgatgaagaa?gatgaccgac?aactgggagc?cctcctgcga?gaagatcatc?cccgtgccca?????840
agcagggcat?cttgaagggc?gacgtgagca?tgtacctgct?gctgaaggac?ggtggccgct?????900
tgcgctgcca?gttcgacacc?gtgtacaagg?ccaagtccgt?gccccgcaag?atgcccgact?????960
ggcacttcat?ccagcacaag?ctgacccgcg?aggaccgcag?cgacgccaag?aaccagaagt????1020
ggcacctgac?cgagcacgcc?atcgcctccg?gctccgcctt?gccctgctct?agatcccgaa????1080
tttccccgat?cgttcaaaca?tttggcaata?aagtttctta?agattgaatc?ctgttgccgg????1140
tcttgcgatg?attatcatat?aatttctgtt?gaattacgtt?aagcatgtaa?taattaacat????1200
gtaatgcatg?acgttattta?tgagatgggt?ttttatgatt?agagtcccgc?aattatacat????1260
ttaatacgcg?atagaaaaca?aaatatagcg?cgcaaactag?gataaattat?cgcgcgcggt????1320
gtcatctatg?ttactagatc?gggaattggg?gaaatttacc?ggtgccgaat?ttccccgatc????1380
cagcttctgg?cagacaaagt?ggcagacata?ctgtcccaca?aatgaagatg?gaatctgtaa????1440
aagaaaacgc?gtgaaataat?gcgtctgaca?aaggttaggt?cggctgcctt?taatcaatac????1500
caaagtggtc?cctaccacga?tggaaaaact?gtgcagtcgg?tttggctttt?tctgacgaac????1560
aaataagatt?cgtggccgac?aggtgggggt?ccaccatgtg?aaggcatctt?cagactccaa????1620
taatggagca?atgacgtaag?ggcttacgaa?ataagtaagg?gtagtttggg?aaatgtccac????1680
tcacccgtca?gtctataaat?acttagcccc?tccctcattg?ttaagggagc?aaggatccat????1740
gaaaaagcct?gaactcaccg?cgacgtctgt?cgagaagttt?ctgatcgaaa?agttcgacag????1800
cgtctccgac?ctgatgcagc?tctcggaggg?cgaagaatct?cgtgctttca?gcttcgatgt????1860
aggagggcgt?ggatatgtcc?tgcgggtaaa?tagctgcgcc?gatggtttct?acaaagatcg????1920
ttatgtttat?cggcactttg?catcggccgc?gctcccgatt?ccggaagtgc?ttgacattgg????1980
ggaattcagc?gagagcctga?cctattgcat?ctcccgccgt?gcacagggtg?tcacgttgca????2040
agacctgcct?gaaaccgaac?tgcccgctgt?tctgcagccg?gtcgcggagg?ccatggatgc????2100
gatcgctgcg?gccgatctta?gccagacgag?cgggttcggc?ccattcggac?cgcaaggaat????2160
cggtcaatac?actacatggc?gtgatttcat?atgcgcgatt?gctgatcccc?atgtgtatca????2220
ctggcaaact?gtgatggacg?acaccgtcag?tgcgtccgtc?gcgcaggctc?tcgatgagct????2280
gatgctttgg?gccgaggact?gccccgaagt?ccggcacctc?gtgcacgcgg?atttcggctc????2340
caacaatgtc?ctgacggaca?atggccgcat?aacagcggtc?attgactgga?gcgaggcgat????2400
gttcggggat?tcccaatacg?aggtcgccaa?catcttcttc?tggaggccgt?ggttggcttg????2460
tatggagcag?cagacgcgct?acttcgagcg?gaggcatccg?gagcttgcag?gatcgccgcg????2520
gctccgggcg?tatatgctcc?gcattggtct?tgaccaactc?tatcagagct?tggttgacgg????2580
caatttcgat?gatgcagctt?gggcgcaggg?tcgatgcgac?gcaatcgtcc?gatccggagc????2640
cgggactgtc?gggcgtacac?aaatcgcccg?cagaagcgcg?gccgtctgga?ccgatggctg????2700
tgtagaagta?ctcgccgata?gtggaaaccg?acgccccagc?actcgtccga?gggcaaagga????2760
atagggatcc?cccgaatttc?cccgatcgtt?caaacatttg?gcaataaagt?ttcttaagat????2820
tgaatcctgt?tgccggtctt?gcgatgatta?tcatataatt?tctgttgaat?tacgttaagc????2880
atgtaataat?taacatgtaa?tgcatgacgt?tatttatgag?atgggttttt?atgattagag????2940
tcccgcaatt?atacatttaa?tacgcgatag?aaaacaaaat?atagcgcgca?aactaggata????3000
aattatcgcg?cgcggtgtca?tctatgttac?tagatcggga?attagcggcc?cgaattcact????3060
ggccgtcgtt?ttacaacgtc?gtgactggga?aaaccctggc?gttacccaac?ttaatcgcct????3120
tgcagcacat?ccccctttcg?ccaggggcgg?ccagcatggc?cgtatccgca?atgtgttatt????3180
aagttgtcta?agcgtcaatt?tgtttacacc?acaatatatc?ctgccaccag?ccagccaaca????3240
gctccccgac?cggcagctcg?gcacaaaatc?accactcgat?acaggcagcc?catcagaatt????3300
aattctcatg?tttgacagct?tatcatcgac?tgcacggtgc?accaatgctt?ctggcgtcag????3360
gcagccatcg?gaagctgtgg?tatggctgtg?caggtcgtaa?atcactgcat?aattcgtgtc????3420
gctcaaggcg?cactcccgtt?ctggataatg?ttttttgcgc?cgacatcata?acggttctgg????3480
caaatattct?gaaatgagct?gttgacaatt?aatcatccgg?ctcgtataat?gtgtggaatt????3540
gtgagcggat?aacaatttca?cacaggaaac?agaccatgag?ggaagcgttg?atcgccgaag????3600
tatcgactca?actatcagag?gtagttggcg?tcatcgagcg?ccatctcgaa?ccgacgttgc????3660
tggccgtaca?tttgtacggc?tccgcagtgg?atggcggcct?gaagccacac?agtgatattg????3720
atttgctggt?tacggtgacc?gtaaggcttg?atgaaacaac?gcggcgagct?ttgatcaacg????3780
accttttgga?aacttcggct?tcccctggag?agagcgagat?tctccgcgct?gtagaagtca????3840
ccattgttgt?gcacgacgac?atcattccgt?ggcgttatcc?agctaagcgc?gaactgcaat????3900
ttggagaatg?gcagcgcaat?gacattcttg?caggtatctt?cgagccagcc?acgatcgaca????3960
ttgatctggc?tatcttgctg?acaaaagcaa?gagaacatag?cgttgccttg?gtaggtccag????4020
cggcggagga?actctttgat?ccggttcctg?aacaggatct?atttgaggcg?ctaaatgaaa????4080
ccttaacgct?atggaactcg?ccgcccgact?gggctggcga?tgagcgaaat?gtagtgctta????4140
cgttgtcccg?catttggtac?agcgcagtaa?ccggcaaaat?cgcgccgaag?gatgtcgctg????4200
ccgactgggc?aatggagcgc?ctgccggccc?agtatcagcc?cgtcatactt?gaagctaggc????4260
aggcttatct?tggacaagaa?gatcgcttgg?cctcgcgcgc?agatcagttg?gaagaatttg????4320
ttcactacgt?gaaaggcgag?atcaccaaag?tagtcggcaa?ataaagctct?agtggatctc????4380
cgtacccggg?gatctggctc?gcggcggacg?cacgacgccg?gggcgagacc?ataggcgatc????4440
tcctaaatca?atagtagctg?taacctcgaa?gcgtttcact?tgtaacaacg?attgagaatt????4500
tttgtcataa?aattgaaata?cttggttcgc?atttttgtca?tccgcggtca?gccgcaattc????4560
tgacgaactg?cccatttagc?tggagatgat?tgtacatcct?tcacgtgaaa?atttctcaag????4620
cgctgtgaac?aagggttcag?attttagatt?gaaaggtgag?ccgttgaaac?acgttcttct????4680
tgtcgatgac?gacgtcgcta?tgcggcatct?tattattgaa?taccttacga?tccacgcctt????4740
caaagtgacc?gcggtagccg?acagcaccca?gttcacaaga?gtactctctt?ccgcgacggt????4800
cgatgtcgtg?gttgttgatc?tagatttagg?tcgtgaagat?gggctcgaga?tcgttcgtaa????4860
tctggcggca?aagtctgata?ttccaatcat?aattatcagt?ggcgaccgcc?ttgaggagac????4920
ggataaagtt?gttgcactcg?agctaggagc?aagtgatttt?atcgctaagc?cgttcagtat????4980
cagagagttt?ctagcacgca?ttcgggttgc?cttgcgcgtg?cgccccaacg?ttgtccgctc????5040
caaagaccga?cggtcttttt?gttttactga?ctggacactt?aatctcaggc?aacgtcgctt????5100
gatgtccgaa?gctggcggtg?aggtgaaact?tacggcaggt?gagttcaatc?ttctcctcgc????5160
gtttttagag?aaaccccgcg?acgttctatc?gcgcgagcaa?cttctcattg?ccagtcgagt????5220
acgcgacgag?gaggtttatg?acaggagtat?agatgttctc?attttgaggc?tgcgccgcaa????5280
acttgaggca?gatccgtcaa?gccctcaact?gataaaaaca?gcaagaggtg?ccggttattt????5340
ctttgacgcg?gacgtgcagg?tttcgcacgg?ggggacgatg?gcagcctgag?ccaattccca????5400
gatccccgag?gaatcggcgt?gagcggtcgc?aaaccatccg?gcccggtaca?aatcggcgcg????5460
gcgctgggtg?atgacctggt?ggagaagttg?aaggccgcgc?aggccgccca?gcggcaacgc????5520
atcgaggcag?aagcacgccc?cggtgaatcg?tggcaagcgg?ccgctgatcg?aatccgcaaa????5580
gaatcccggc?aaccgccggc?agccggtgcg?ccgtcgatta?ggaagccgcc?caagggcgac????5640
gagcaaccag?attttttcgt?tccgatgctc?tatgacgtgg?gcacccgcga?tagtcgcagc????5700
atcatggacg?tggccgtttt?ccgtctgtcg?aagcgtgacc?gacgagctgg?cgaggtgatc????5760
cgctacgagc?ttccagacgg?gcacgtagag?gtttccgcag?ggccggccgg?catggccagt????5820
gtgtgggatt?acgacctggt?actgatggcg?gtttcccatc?taaccgaatc?catgaaccga????5880
taccgggaag?ggaagggaga?caagcccggc?cgcgtgttcc?gtccacacgt?tgcggacgta????5940
ctcaagttct?gccggcgagc?cgatggcgga?aagcagaaag?acgacctggt?agaaacctgc????6000
attcggttaa?acaccacgca?cgttgccatg?cagcgtacga?agaaggccaa?gaacggccgc????6060
ctggtgacgg?tatccgaggg?tgaagccttg?attagccgct?acaagatcgt?aaagagcgaa????6120
accgggcggc?cggagtacat?cgagatcgag?ctagctgatt?ggatgtaccg?cgagatcaca????6180
gaaggcaaga?acccggacgt?gctgacggtt?caccccgatt?actttttgat?cgatcccggc????6240
atcggccgtt?ttctctaccg?cctggcacgc?cgcgccgcag?gcaaggcaga?agccagatgg????6300
ttgttcaaga?cgatctacga?acgcagtggc?agcgccggag?agttcaagaa?gttctgtttc????6360
accgtgcgca?agctgatcgg?gtcaaatgac?ctgccggagt?acgatttgaa?ggaggaggcg????6420
gggcaggctg?gcccgatcct?agtcatgcgc?taccgcaacc?tgatcgaggg?cgaagcatcc????6480
gccggttcct?aatgtacgga?gcagatgcta?gggcaaattg?ccctagcagg?ggaaaaaggt????6540
cgaaaaggtc?tctttcctgt?ggatagcacg?tacattggga?acccaaagcc?gtacattggg????6600
aaccggaacc?cgtacattgg?gaacccaaag?ccgtacattg?ggaaccggtc?acacatgtaa????6660
gtgactgata?taaaagagaa?aaaaggcgat?ttttccgcct?aaaactcttt?aaaacttatt????6720
aaaactctta?aaacccgcct?ggcctgtgca?taactgtctg?gccagcgcac?agccgaagag????6780
ctgcaaaaag?cgcctaccct?tcggtcgctg?cgctccctac?gccccgccgc?ttcgcgtcgg????6840
cctatcgcgg?ccgctggccg?ctcaaaaatg?gctggcctac?ggccaggcaa?tctaccaggg????6900
cgcggacaag?ccgcgccgtc?gccactcgac?cgccggcgct?gaggtctgcc?tcgtgaagaa????6960
ggtgttgctg?actcatacca?ggcctgaatc?gccccatcat?ccagccagaa?agtgagggag????7020
ccacggttga?tgagagcttt?gttgtaggtg?gaccagttgg?tgattttgaa?cttttgcttt????7080
gccacggaac?ggtctgcgtt?gtcgggaaga?tgcgtgatct?gatccttcaa?ctcagcaaaa????7140
gttcgattta?ttcaacaaag?ccgccgtccc?gtcaagtcag?cgtaatgctc?tgccagtgtt????7200
acaaccaatt?aaccaattct?gattagaaaa?actcatcgag?catcaaatga?aactgcaatt????7260
tattcatatc?aggattatca?ataccatatt?tttgaaaaag?ccgtttctgt?aatgaaggag????7320
aaaactcacc?gaggcagttc?cataggatgg?caagatcctg?gtatcggtct?gcgattccga????7380
ctcgtccaac?atcaatacaa?cctattaatt?tcccctcgtc?aaaaataagg?ttatcaagtg????7440
agaaatcacc?atgagtgacg?actgaatccg?gtgagaatgg?caaaagctct?gcattaatga????7500
atcggccaac?gcgcggggag?aggcggtttg?cgtattgggc?gctcttccgc?ttcctcgctc????7560
actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca?ctcaaaggcg????7620
gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg?agcaaaaggc????7680
cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc????7740
ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga????7800
ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc????7860
ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcat????7920
agctcacgct?gtaggtatct?cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg????7980
cacgaacccc?ccgttcagcc?cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc????8040
aacccggtaa?gacacgactt?atcgccactg?gcagcagcca?ctggtaacag?gattagcaga????8100
gcgaggtatg?taggcggtgc?tacagagttc?ttgaagtggt?ggcctaacta?cggctacact????8160
agaagaacag?tatttggtat?ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt????8220
ggtagctctt?gatccggcaa?acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag????8280
cagcagatta?cgcgcagaaa?aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg????8340
tctgacgctc?agtggaacga?aaactcacgt?taagggattt?tggtcatgag?attatcaaaa????8400
aggatcttca?cctagatcct?tttgatccgg?aattaattcc?tgtggttggc?atgcacatac????8460
aaatggacga?acggataaac?cttttcacgc?ccttttaaat?atccgattat?tctaataaac????8520
gctcttttct?cttaggttta?cccgccaata?tatcctgtca?aacactgata?gtttaaactg????8580
aaggcgggaa?acgacaatct?gatcatgagc?ggagaattaa?gggagtcacg?ttatgacccc????8640
cgccgatgac?gcgggacaag?ccgttttacg?tttggaactg?acagaaccgc?aacgctgcag????8700
gaattggccg?cagcggccat?ttaaatcaat?tgggcgcgcc?gaattcgagc?tcggtac???????8757
Claims (22)
1. the method for a soybean transformation cell or tissue comprises:
(a) prepare explant by following steps from soybean seeds:
(i) from said soybean seeds, remove hypocotyl;
(ii) remove a cotyledon and contiguous axillalry bud thereof, keep investing remaining epicotyledonary primary leaf; And
(iii) remove a part of primary leaf, produce the primary leaf base portion thus from said reservation cotyledon; And
(b) said explant and the Agrobacterium that contains the purpose nucleic acid at least one genome of waiting to be incorporated into one or more soya cells are cultivated altogether.
2. the method for claim 1 also is included in the bud of cultivating at least one formation in the substratum that contains selective agent.
3. the method for claim 2, wherein said at least one purpose nucleic acid comprises selectable marker gene.
4. the method for claim 3, wherein said selectable marker gene is a Phophomannose isomerase gene.
5. the method for claim 4, wherein said selective agent is a seminose.
6. the method for claim 4, wherein cultivating altogether with said Agrobacterium is to carry out under the situation that seminose exists.
7. the method for claim 2 also comprises from said primary leaf base portion induced bud forming.
8. the method for claim 7, wherein to form be by inducing and cultivate said primary leaf base portion inductive in the substratum of hormone containing bud to bud.
9. the method for claim 8, wherein said bud induce hormone to comprise at least a in growth hormone, phytokinin and the Plant hormones regulators,gibberellins.
10. the method for claim 9, wherein said growth hormone is selected from the group of being made up of IAA, NAA and IBA.
11. the method for claim 9, wherein said phytokinin is selected from the group of being made up of benzyladenine (BAP), thidiazuron, kinetin and isopentenyl gland purine.
12. the method for claim 7, wherein bud forms to induce and comprises individual or a plurality of epicotyledonary primary meristem, secondary meristem and the axillary meristems of being attached to of removal.
13. the method for claim 7 also comprises the bud of selecting conversion.
14. the method for claim 13 comprises that also the conversion shoot regeneration that will select is a soybean plants.
15. the process of claim 1 wherein that said soybean seeds is a mature seed.
16. the process of claim 1 wherein that said soybean seeds is an immature seed.
17. the process of claim 1 wherein that said soybean seeds is a chitting piece.
18. a method of producing the soybean plants of stable conversion comprises:
(a) prepare explant by following steps from soybean seeds:
(i) from said soybean seeds, remove hypocotyl;
(ii) remove a cotyledon and contiguous axillalry bud thereof, keep attached to remaining epicotyledonary primary leaf; And
(iii) remove the part of each primary leaf, thereby produce a pair of primary leaf base portion from said reservation cotyledon;
(b) with said explant with contain the Agrobacterium that remains to be incorporated into the purpose nucleic acid in the soya cells genome and cultivate altogether;
(c) from the formation of each primary leaf base portion induced bud;
(d) in containing the substratum of selective agent, cultivate the bud of a formation at least.
(e) bud of selection conversion; And
(f) the conversion shoot regeneration that will select is a soybean plants.
19. from the soya cells that transforms according to the method for claim 1 or the genetically engineered soybean plant of tissue regeneration.
20. the transgenic seed that the transgenic plant of claim 19 produce.
21. from the soya cells that transforms according to claim 18 method or the genetically engineered soybean plant of tissue regeneration.
22. the transgenic seed that the transgenic plant of claim 21 produce.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US39056202P | 2002-06-22 | 2002-06-22 | |
| US60/390,562 | 2002-06-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1668744A true CN1668744A (en) | 2005-09-14 |
Family
ID=30000570
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN03816844.8A Pending CN1668744A (en) | 2002-06-22 | 2003-06-17 | Method of transforming soybean |
Country Status (11)
| Country | Link |
|---|---|
| US (2) | US20040034889A1 (en) |
| EP (1) | EP1517991A4 (en) |
| JP (1) | JP2005535312A (en) |
| CN (1) | CN1668744A (en) |
| AR (1) | AR039721A1 (en) |
| AU (1) | AU2003279185B2 (en) |
| BR (1) | BR0312005A (en) |
| CA (1) | CA2490154A1 (en) |
| NZ (1) | NZ537237A (en) |
| WO (1) | WO2004000006A2 (en) |
| ZA (1) | ZA200410134B (en) |
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| CN102329816A (en) * | 2011-06-15 | 2012-01-25 | 北京未名凯拓作物设计中心有限公司 | Agrobacterium-mediated method for transferring soybean from top end of epicotyl of longitudinally-cut seedling |
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| US6664109B2 (en) * | 1985-01-17 | 2003-12-16 | Calgene Llc | Transformation system with Ti or Ri plasmid |
| US5169770A (en) * | 1987-12-21 | 1992-12-08 | The University Of Toledo | Agrobacterium mediated transformation of germinating plant seeds |
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| EP0652965A1 (en) * | 1992-07-27 | 1995-05-17 | Pioneer Hi-Bred International, Inc. | An improved method of agrobacterium-mediated transformation of cultured soybean cells |
| GB9307408D0 (en) * | 1993-04-08 | 1993-06-02 | Danisco | Transgenic plants |
| US5959179A (en) * | 1996-03-13 | 1999-09-28 | Monsanto Company | Method for transforming soybeans |
| US5968830A (en) * | 1997-03-28 | 1999-10-19 | Mississippi State University | Soybean transformation and regeneration methods |
| AU6240899A (en) * | 1998-08-31 | 2000-03-21 | Monsanto Technology Llc | Transgene assay using stable agrobacterium rhizogenes transformation |
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| EP1311696B1 (en) * | 2000-08-11 | 2012-07-25 | Syngenta Participations AG | Methods for stable transformation of plants |
| US20030046733A1 (en) * | 2001-09-06 | 2003-03-06 | Dias Kalyani Mallika | Transformation of soybeans |
-
2003
- 2003-06-17 AU AU2003279185A patent/AU2003279185B2/en not_active Ceased
- 2003-06-17 JP JP2004515886A patent/JP2005535312A/en active Pending
- 2003-06-17 US US10/463,074 patent/US20040034889A1/en not_active Abandoned
- 2003-06-17 CN CN03816844.8A patent/CN1668744A/en active Pending
- 2003-06-17 NZ NZ537237A patent/NZ537237A/en unknown
- 2003-06-17 CA CA002490154A patent/CA2490154A1/en not_active Abandoned
- 2003-06-17 US US10/517,039 patent/US20060260012A1/en not_active Abandoned
- 2003-06-17 WO PCT/US2003/019212 patent/WO2004000006A2/en active Application Filing
- 2003-06-17 EP EP03742051A patent/EP1517991A4/en not_active Withdrawn
- 2003-06-17 BR BR0312005-8A patent/BR0312005A/en not_active IP Right Cessation
- 2003-06-20 AR ARP030102206A patent/AR039721A1/en unknown
-
2004
- 2004-12-15 ZA ZA200410134A patent/ZA200410134B/en unknown
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Also Published As
| Publication number | Publication date |
|---|---|
| AR039721A1 (en) | 2005-03-09 |
| ZA200410134B (en) | 2005-11-22 |
| AU2003279185A1 (en) | 2004-01-06 |
| JP2005535312A (en) | 2005-11-24 |
| WO2004000006A3 (en) | 2004-05-13 |
| WO2004000006A2 (en) | 2003-12-31 |
| CA2490154A1 (en) | 2003-12-31 |
| AU2003279185B2 (en) | 2008-05-29 |
| BR0312005A (en) | 2005-08-16 |
| US20060260012A1 (en) | 2006-11-16 |
| EP1517991A4 (en) | 2006-03-29 |
| EP1517991A2 (en) | 2005-03-30 |
| US20040034889A1 (en) | 2004-02-19 |
| NZ537237A (en) | 2008-01-31 |
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